Poster Presentations Session 1: Environment and Health

Transcription

Acta Biochim Biophys Sin 2010, 42: i19 – i66 | ª The Author 2010. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmq053.
Poster Presentations
Session 1: Environment and Health
Po01. Molecular signaling involved in low
doses of arsenic-promoted cell
proliferation
Youhong Liu 1, Con Sullivan 1, Geying Fang 1,
Allison Cox 1, and Xiong Li 1,2 *
Maine Institute for Human Genetics and Health
Graduate School of Biomedical Science, University of Maine,
246 Sylvan Road, Bangor, ME, USA
*Correspondence address. Tel: þ1-207-973-9310;
Fax: þ1-207-973-9307; E-mail: [email protected]
2
Arsenic trioxide (ATO) has been approved as a first-line
anticancer agent for acute promyelocytic leukemia, and
induces apoptosis in other solid cancer cell lines including
breast cancer cells. However, as one type of arsenites existing in drinking water, and as a raw material used for the
manufacturing of wood preservatives, insecticides and herbicides, ATO might induce carcinogenesis at low doses for
a long duration exposure. Low dose ATO significantly promotes cell proliferation. We investigated the molecular signaling involved in low doses of arsenic-induced cell
proliferation.
The cell proliferation and cell cycle progression were
tested when MCF10A, a non-tumorigenic breast epithelial
cell line, was exposed at low doses of ATO. The involved
cell cycle-associated genes were screened using a Human
Cell Cycle Tox and Cancer StellARayTM qPCR Array. The
alterations of target genes were confirmed by RT-PCR and
Western blot. In addition, the production of reactive oxidative species (ROS) was tested by flow cytometry, and
the activation of p38 MAPK, Akt and ErK1/2 was monitored using the phosphorylated antibodies by Western blot.
The interactions between the activation of p38 MAPK, Akt
and ErK1/2 pathways and the expression of cell cycle
genes were investigated by using the small molecular
pathway inhibitors and the results were confirmed when
these pathways were inhibited by siRNAs.
ATO (0.01-1 mM) significantly increased cell proliferation and promoted cell cycle progression from G1 to S/G2
phases at 24 h after exposure. The expression of 14 out of
96 cell cycle associated genes significantly increased, and
Po02. Combined effects of serum trace
metals and polymorphisms of CYP1A1 or
GSTM1 on non-small cell lung cancer, a
hospital based case-control study in
China
Yongtang Jin 1, Chenye Zhang 1, Heyun Xu 2, Shaoli Xue 3,
Yasong Wang 4, Yong Hou 4, Yunming Kong 4, and
Yingchun Xu 5
1
Department of Environmental Medicine or Institute of
Environmental Medicine, Zhejiang University School of Medicine,
Zhejiang University, Hangzhou, China
2
Department of Cardiothoracic Surgery, Sir Run Run Shaw Hospital,
Zhejiang University School of Medicine, Zhejiang University,
Hangzhou, China
3
Department of Biotechnology, Anhui Medical University, Hefei,
China
4
School of Public Health, Anhui Medical University, Hefei, China
5
Institute of Pharmacology, Zhejiang University School of
Pharmacology, Zhejiang University, Hangzhou, China
E-mail: [email protected]
There is limited information for the effects of serum
trace elements and genetic polymorphisms about lung
cancer. This study examined the association of trace
elements level in serum with genetic polymorphisms with
Acta Biochim Biophys Sin (2010) | Volume 42 | Supplement 1 | Page i19
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seven genes including Cell division cycle 6 (CDC6) and
cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low dose ATO steadily
increased gene transcription and protein levels of both
CDC6 and cyclin D1 in a dose- and time-dependent
manner. Low dose ATO produced reactive oxygen species
(ROS), and activated p38 MAPK, Akt and ErK1/2 pathways at different time points within 60 min. The inhibition
of activation of p38 MAPK, Akt and ErK1/2 by both small
molecular inhibitors and siRNAs decreased ATO-increased
expression of CDC6 protein. Inhibition of activation of Akt
and ErK1/2, but not p38 MAPK, decreased ATO-increased
expression of cyclin D1 protein.
This study reports for the first time that the activation of
p38 MAPK/ Akt /ErK1/2 are required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO-induced
cell proliferation and cell cycle modulation from G1 to S
phase.
Abstracts
Po03. Pathways affected by Fe2O3
nanoparticles with microRNA expression
profiling
Shuchun Li, Nan Ye, Yuhua Qi, Gaofeng Liang, and
Zhongdang Xiao *
State Key Laboratory of Bioelectronics, School of Biological Science
& Medical Engineering, Southeast University, Nanjing, China
*Correspondence address. Tel: þ86-25-83790820; Fax: þ86-2583790820; E-mail: [email protected]
Nanomaterials have received considerable attention in
recent years because of their unique properties and diverse
applications in biotechnology and life sciences. At the same
time, the evaluation of their potential adverse health effects
has also become urgent. Many studies have been performed
to investigate the biological effects of nanomaterials on
DNA, RNA, and protein. Recent studies have revealed that
microRNA could regulate the gene expression at the translation level. MicroRNAs have been found to be involved in
many important physiological and/or pathological processes, such as cell differentiation, apoptosis and tumorigenesis. This study investigated miRNA expression profiling in
NIH/3T3 cells exposed to Fe2O3 nanoparticles. The doseand time-dependent effects of Fe2O3 nanoparticles exposure
on microRNAs were detected with SOLiD sequence and
quantitative real-time PCR (qRT-PCR). Subsequently, cellular pathways affected by the significantly regulated
microRNAs were analyzed with DIANA-mirPath. Both
SOLiD and qRT-PCR detected dramatically changed
microRNA expression profiling after Fe203 nanoparticle
exposure. Cellular pathways, such as MAPK signaling
pathway, focal adhesion, and regulation of actin cytoskeleton were significantly affected by two group microRNAs.
The present study thus provides a feasible method that combined SOLiD sequencing with qRT-PCR, to detect
microRNA profiling. In addition, analysis with union of all
microRNAs was more reliable than separate microRNA. By
combining high-through microRNA expression profiling
and bioinformatics assay, we are possible to investigate the
global biological effects of Fe2O3 nanoparticles.
Po04. Psychometric study on the
relationship between MMORPG
addiction and motivations
Ge Qian *
Humanities College, Shanghai University of Finance and Economics,
Shanghai, China
*Correspondence address. Tel: þ86-21-58312236; Fax: þ86-2165904720; E-mail: [email protected]
These are a few of the myriad of virtual phenomena that
occur every day in online digital constructs known as
Massively Multi-User Online Role-Playing Games
(MMORPG), and millions of users participate fanatically
in these online environments. This study is to examine the
relationship between the motivations and MMORPG addiction. Data were gathered from Shanghai University of
Finance and Economics. Questionnaires were distributed to
400 undergraduate students, 341 pieces of questionnaire
were back and the response rate was 85.3%.
The questionnaire used in this research was designed by
Nick Yee (2007), but is modified and translated to
Chinese. Items were used to gather basic information such
as hours of usage per week as an indicator to estimate the
extent of addiction, and explore the motivations to play
MMORPG, which included achievement seeking, sociability, immersion and sensation seeking.
Results are shown in the two tables.
Unlike the previous research (Ann & Randall, 2007), it
is found in this study that sociability is not a predictor of
MMORPG addiction, but immersion is.
the development of lung cancer. Based on a hospital-based
case-control study, the epidemiological questionnaires were
completed by face to face interview, and the gene polymorphisms were tested by RFLP-PCR, and serum trace
metals were measured by atomic absorption spectrophotometer. The data were then analyzed by the logistic regressive models. It was found that high serum copper level
(.1500 ng/ml) or serum copper/zinc ratio (.1) was the
risk factors for NSCLC (OR ¼ 3.10, 11.03, respectively),
but the ORs of the higher serum Zn (.1200 ng/ml), Se
(.50 ng/ml) or Cr3þ (.600 ng/ml) for non-small cell
lung cancer (NSCLC) were all significantly less than 0.20
(all P , 0.01), indicating strong protection against NSCLC.
While the OR of CYP 1A1 variants carriers with a higher
serum Cu or Cu/Zn ratio level was around 3.38 and 12.59,
respectively, the risk of CYP1A1 variants carriers with a
higher serum Zn is 0.18, Se 0.04 or Cr3þ 0.28. Similarly,
compared with the carriers of GSTM1 power with a lower
serum Zn, Se or Cr3þ, the OR of the carriers of GSTM1
null with a higher serum Zn, Se and Cr3þ was 0.16, 0.07
and 0.26, respectively, highlighting the protection against
NSCLC. Together, our findings suggested that CYP1A1 or
GSTM1 variants may significantly modify the associations
between level of serum trace metals (Cu, Zn, Se or Cr) and
NSCLC, indicating the intriguing pathogenesis of lung
cancer.
Abstracts
Table 1 Correlations between MMORPG addiction and motivations
(*P< 50.01; **P< 50.001; n5341)
Motivation
Addiction
Pearson correlation P value
Achievement
Sociability
Immersion
Sensation seeking
0.415**
0.439**
0.483**
0.201*
0.000
0.000
0.000
0.001
Po06. The combined toxicity of dibutyl
Phthalate and benzo(a)pyrene in primary
cultured rat sertoli cells in vitro
Zhiqun Qiu 1,2 , Jiaohua Luo 2, Ji’an Chen 2, Lan Yang 2,
Weiqun Shu 2 *, and Jia Cao 1*
1
Table 2 Regression analysis of motivations on MMORPG addiction
(**P < 50.001; n5341)
Predictors
Addiction
Motivation
Achievement
Sociability
Immersion
Sensation seeking
b
P value
20.015
n.s.
0.142
n.s.
0.303
0.001**
0.094
n.s.
Ying Zhao 1, Songping Luo 1, Ruqiang Ou 2, and Jie Gao 1
1
Department of gynecology, First College of Medicine of Guangzhou
University of Traditional Chinese Medicine, Guangzhou 510405, China
2
Family Planning Research Institute of Guangdong Province,
Guangzhou 510600, China
To study whether Chinese herbal medicine could interfere
with the toxic effects of deltamethrin (DM) on reproduction,
pregnant rats were divided into five groups. Groups 1-4
were orally fed 1/20 LD50 (6.93 mg/kg) DM daily from Day
1 of pregnancy to day 15. Physiological saline was given
4 h after being poisoned in Group 1. The Chinese herbal
formula Zhuyun III was given 4 times, 2 times, or 1 time
daily for Group 2, 3, and 4, respectively. Group 5 was blank
control. Half of the pregnant rats were sacrificed on day 15.
The rest were kept until labour. The general abortion rate,
levels of estradiol, prostaglandin, and Th1/Th2 ratio were
examined. The abortion rate was significantly higher in
Group 1 compared with other groups. The progesterone
level was also low in Group 1, and the Th1/Th2 ratio
showed tendency to Th1 type. Zhuyun III is a formula to
enhance the function of kidney and spleen. On the other
hand, treated with Formula Zhuyun III, the toxic effects of
DM on abortion rate, level of progesterone, Th1/Th2 ratio
were significantly alleviated. DM has toxicity on reproduction, which can lead to abortion. Formula Zhuyun III could
improve the above condition and relief DM toxicity.
Humans are exposed to a vast array of chemical mixtures.
Phthalates (PAEs) and polycyclic aromatic hydrocarbons
(PAHs) are the most abundant POPs in the environment.
Although a previous study showed that both PAEs and
PAHs selectively target Sertoli cells, there are few published
reports about the combined toxicity of PAEs and PAHs. To
determine the combined toxicity of PAEs and PAHs in
Sertoli cells, we used dibutyl phthalate (DBP) and benzo(a)pyrene (BaP) as model chemicals to investigate the effects
of single or combined treatments on cultured primary rat
Sertoli cells. Sertoli cells prepared from 18- to 21-day-old
Sprague-Dawley rats were treated with 1% dimethyl sulfoxide (vehicle); 0.1, 1, 10, 100, and 500 mg/ml DBP; 0.01,
0.1, 1, 10, and 100 mg/ml BaP; or DBP þ BaP (combined
from low to high concentrations) for 12 h and 24 h for MTT
assays; or with 1, 10, and 100 mg/ml DBP; 0.1, 1, and
10 mg/ml BaP; and DBP þ BaP (combined from low to
high doses) for 12 h and 24 h for evaluation of ultramicrostructure, lactate, inhibin B and transferrin. Sertoli cell proliferation was stimulated by 500 mg/ml DBP and 100 mg/ml
DBP þ 10 mg/ml BaP in a concentration-dependent manner
(P , 0.05). Lactate secretion was stimulated by 100 mg/ml
DBP and 100 mg/ml DBP þ 10 mg/ml BaP (P , 0.01), but
was inhibited by BaP (P , 0.05). The expression of INH B
mRNA was decreased by BaP treatment (all three groups at
12 h) and increased by 100 mg/ml DBP at 12 h and 24 h
(P , 0.05), and Tf mRNA levels in Sertoli cells were
increased by DBP, BaP and DBP þ BaP at 24 h. DBP
stimulated Sertoli cell proliferation. In addition, lactate
secretion was stimulated by DBP, but was inhibited by BaP.
Both DBP and BaP can disturb active protein secretion, and
the main combined toxic effect of DBP and BaP is antagonistic. Because the combined effects of DBP and BaP are
complicated, the mechanism of action should be further
examined in future study.
Po05. Experimental study of Chinese
herbal medicine affecting pregnant rats
being poisoned by deltamethrin (DM)
Department of Hygenic Toxicology, School of Military Preventive
Medicine, Key Lab of Medical Protection for Electromagnetic
Radiation, Ministry of Education of China, Third Military Medical
University, Chongqing, China
2
Department of Environmental Hygiene, School of Military Preventive
Medicine, Third Military Medical University, Chongqing, China
Abstracts
Po07. Benzo[a]pyrene induces complex
H2AX phosphorylation patterns by
multiple kinases including ATM, ATR,
and DNA-PK
Chunlan Yan 1, Guanglin Zhang 1, Tie’er Gan 1,
Qunli Zeng 2, Zhengping Shao 1, Penelope
J. Duerksen-Hughes 3, and Jun YANG 1 *
1
Department of Toxicology, Zhejiang University School of Public
Health, Hangzhou, China
2
Department of Environmental and Occupational Health, Zhejiang
University School of Public Health, Hangzhou, China
3
Department of Biochemistry and Microbiology, Loma Linda
University School of Medicine, Loma Linda, CA, USA
*Correspondence address. Tel/Fax: þ86-571-88208140;
Han Wu 1, Yihua Wu 2, Jing Lu 2, Jun Yang 2*, and Juan Ye 1*
1
Department of Ophthalmology, the Second Affiliated Hospital,
2
E-mail: [email protected] (J. Y.)
Tel/Fax: þ86-571-87783897; E-mail: [email protected] (J. Y.)
Ethylenediaminetetraacetic acid disodium salt (EDTA) is
commonly used in ophthalmic solutions as a buffering agent.
It has been reported that EDTA could promote corneal drug
penetration presumably as the consequence of ultrastructural
changes induced in the corneal epithelium. However, it is not
clear whether EDTA could cause DNA damage in human
corneal epithelial cell. Therefore, using phosphorylated
H2AX (gH2AX) foci formation as an indicator for DNA
damage, we evaluated the genotoxic effect of EDTA in
immortalized human corneal epithelial cells (HCEs). It was
found that EDTA exhibits no significant cytotoxic effects on
HCEs. However, gH2AX foci formation was observed by
EDTA treatment in a time- and dose-dependent manner with
all the concentrations tested (0.00001%, 0.0001% 0.001%
and 0.01%; w/v). Twenty-four hours post-treatment, the
number of gH2AX foci decreased significantly, indicating a
possible repair of the DNA damage induced by EDTA. In
addition, EDTA treatment leads to elevated intracellular reactive oxygen species (ROS) levels. Taken together, these
results suggest EDTA could induce DNA damage and ROS
generation in HCEs, and thus the utilization of EDTA in eye
drop should be carefully monitored.
Po09. Evaluation of the genotoxic effects
of melamine and cyanuric acid using
gH2AX foci formation as an indicator
Jing Lu 1,2, Qian Tang 1,2, Yihua Wu 2, Guanglin Zhang 2,
Xinqiang Zhu 2, and Jun Yang 1,2*
1
Zhejiang-California International Nanosystems Institute, Hangzhou,
China
2
The incident of melamine-contaminated pet food in
North America and melamine-contaminated milk in China
H2AX is phosphorylated (gH2AX) by members of the
phosphatidylinositol 3-kinase (PI3K) family, including
ataxia telangiectasia-mutated (ATM), ATM- and
Rad3-related (ATR) and DNA-PK in response to DNA
damage. While it has been reported that benzo[a]pyrene
(BaP) cannot induce gH2AX alone in several cell lines, we
have shown that BaP alone could induce gH2AX in human
amnion FL cells. Thus, we further examined the ability of
BaP to induce gH2AX in different cell systems. It was
shown that BaP induced gH2AX in HeLa cells in a timeand dose-dependent manner. BaP also induced gH2AX in
ATM2/2 mouse fibroblasts, DNA-PKcs2/2 mouse fibroblasts, and a genetically modified human osteosarcoma
U2OS cell line. PI3K inhibitors caffeine and wortmannin
were then used in an effort to identify the kinase(s) responsible for BaP-induced gH2AX. Unexpectedly, in
BaP-treated HeLa cells, caffeine pretreatment did not inhibit
but rather increased gH2AX level. On the other hand, caffeine or wortmannin can inhibit BaP-induced gH2AX in
U2OS, DNA-PKcs2/2 or ATM2/2 cells. Taken together,
these data suggest that BaP alone can induce H2AX phosphorylation in certain cells, and members of the PI3K
family, including ATM, ATR, and DNA-PK can participate
in the phosphorylation of H2AX in the various cell types.
[This work was supported by grants from the National
Natural Science Foundation of China (Nos. 30771826,
30872140); Ministry of Science and Technology, China
(No. 2009DFB30390); Department of Science and
Technology, Zhejiang Province (No. 2009C11122).]
Po08. Ethylenediaminetetraacetic acid
disodium salt induces the phosphorylation
of histone variant H2AX and generation
of reactive oxygen species in human
corneal epithelial cells
Abstracts
Po10. Vascular endothelial toxicity
induced by silica nanoparticle in vitro
Jun Zhang 1, Yifan Zheng 1, Jun Yang 1,2, and
Xinqiang Zhu 1 *
1
2
China
Once nanoparticle translocated into the circulation, it may
directly contact with vascular endothelial cells, and then
induces toxic effects. In the present study, we investigated
the possible vascular toxic effects of silica nanoparticles
(SiO2 NPs) using human umbilical vein endothelial cells
(HUVECs) as the model system. TEM analysis showed that
SiO2 NPs were observed in the form of randomly dispersed
agglomerates in the cytoplasm. SiO2 NPs at concentrations
of 50 mg/ml and 100 mg/ml significantly induced HUVECs
apoptosis (17.9% and 15.3%, respectively), promoted the
formation of gH2AX foci, and increased sICAM-1
secretion. Furthermore, we found that intracellular oxidative
stress biomarker HO-1 and 8-OHdG formation was significantly elevated followed by the increase of ROS in SiO2
NPs treated cells. We also observed that SiO2 NPs significantly promoted total NO generation, and eNOS may play
an important role in the toxicities of SiO2 NPs. In conclusion, our results demonstrated the toxic effects of SiO2
NPs on HUVECs, and these effects might be partially due
to the enhanced ROS generation and eNOS function. [This
work was supported by grants from the National Natural
Science Foundation of China (No. 30771824, 30800923);
Ministry of Science and Technology, China (No.
2009DFB30390); Natural Science Foundation of Zhejiang
Province (No. Y206537); Department of Science and
Technology, Zhejiang Province (No. 2009C11122); Science
Foundation
of
Chinese
University
(No. 5A7000-172210121).]
Po11. Cardiovascular toxicity of silica
nanoparticles in rats
Jun Zhang 1, Yifan Zheng 1, Jun Yang 1,2, and
Xinqiang Zhu 1 *
1
2
China
In view of the fact that nanomaterials may play an important role in the cardiovascular toxicity, we examined the cardiovascular toxicity of silica nanoparticles (SiO2 NPs) in rats.
The effects of three concentrations of SiO2 NPs (2.5 mg/kg,
5 mg/kg and 10 mg/kg), and 10 mg/ kg SiO2 micron particles
(micro-SiO2) were studied. We found that only the highest
dosage of SiO2 NPs (10 mg/kg) treatment decreased the systolic pressure (SP) and the diastolic pressure (DP) after 2 h
and 4 h treatment compared with controls, and there was no
difference in heart rate among all groups. Similarly, only 1 h,
2 h and 4 h after the administration of the highest dosage of
SiO2 NPs remarkably induced the formation time of thrombus. Although 10 mg/kg micro-SiO2 and all three concentrations of SiO2 NPs could not affect blood platelet count
after 24 h treatment, 5 mg/kg and 10 mg/kg of SiO2 NPs
groups could significantly increased the platelet aggregation
in rats. In conclusion, SiO2 NPs showed cardiovascular toxicity in vivo, with 5 mg/kg and 10 mg/kg of the SiO2 NPs
groups could significantly decrease SP, DP, formation time
of thrombus, and platelet aggregation rate in rats. [This work
was supported by grants from the National Natural Science
Foundation of China (No. 30771824, 30800923); Ministry of
had revived the study for the toxic effects of melamine,
and it has been suggested that the melamine-cyanuric acid
(CA) complex may contribute to the toxic effects in
affected individuals. However, it is not clear whether this
complex has any genotoxic effects. Therefore, in the
present study, using gammaH2AX foci formation as an
indicator for DNA damage, we evaluated the genotoxic
effects of melamine, CA or melamine-CA complex in
human embryonic kidney 293 cells. It was found that
during a 24-h time frame neither melamine nor CA showed
significant toxic effects to 293 cells at 0.5 mg/ml, while
higher concentrations (1 and 5 mg/ml) showed certain
degree of toxicity. A different combination of melamine
and CA [100:1, 10:1, 1:1, 1:10, and 1:100 for
melamine:CA (v:v) at 0.5 mg/ml] also showed cytotoxicity
to various extents. On the other hand, while neither melamine nor CA alone induced gammaH2AX foci formation,
melamine:CA at 100:1 ratio could induce gammaH2AX
foci. Taken together, these data indicate that when melamine and CA are present at a certain ratio, the complex
could induce DNA damage, and thus the long-term effect
in those children who consumed melamine-contaminated
milk should be carefully monitored. [This work was supported by grants from the National Natural Science
Foundation of China (Nos. 30771826, 30872140); Ministry
of Science and Technology, China (No. 2009DFB30390);
Department of Science and Technology, Zhejiang Province
(No. 2009C11122).]
Abstracts
Science and Technology, China (No. 2009DFB30390);
Natural Science Foundation of Zhejiang Province (No.
Y206537); Department of Science and Technology, Zhejiang
Province (No. 2009C11122); Science Foundation of Chinese
University (No. 5A7000-172210121).]
Po12. The toxic effects of single-walled
carbon nanotubes (SWCNTs) on the
structure and function of blood-brain
barrier in vitro
Jun Zhang 1, Qiaohui Wei 1, Yifan Zheng 1, Jun Yang 1,2,
and Xinqiang Zhu 1 *
1
To study the toxic effects of single-walled carbon nanotubes (SWCNTs) on the structure and function of blood-brain
barrier, we established the mouse brain microvascular endothelial cells (bEnd.3) and primary mouse astrocytes (As)
co-culture in vitro blood-brain barrier model. Our results
showed that 50 mg/ml and 200 mg/ml non-modified short
SWCNTs and 200 mg/ml hydroxy-modified short SWCNTs,
carboxyl-modified short SWCNTs and the carboxylmodified long SWCNTs treatments could significantly
decrease the TEER value and tight junction related protein
claudin-1 expression in blood-brain barrier model, and
increase the permeability of HRP. It was further shown that
SWCNTs significantly induced NO and ROS generation in
bEND.3 cells, and increased Ca2þ inflow in a short period of
time (3.5 min). However, no obvious uptake of SWCNTs
in bEND.3 cells was found using TEM and fluorescence
microscopy. Western blotting analysis also showed no
increase of the endocytosis-mediated protein caveolin-1.
These results indicated that non-modified, hydroxyl-modified
and carboxyl-modified SWCNTs could damage the bloodbrain barrier in in vitro assays, and there was no significant
difference in the toxic effects between short SWCNTs and
long SWCNTs. The generation of second messenger NO and
ROS may contribute to the damage of the blood-brain
barrier. [This work was supported by grants from the
National Natural Science Foundation of China (No.
30771824, 30800923); Ministry of Science and Technology,
China (No. 2009DFB30390); Natural Science Foundation of
Zhejiang Province (No. Y206537); Department of Science
and Technology, Zhejiang Province (No. 2009C11122);
Science Foundation of Chinese University (No.
5A7000-172210121).]
Linfeng Chi 1,2, Zhanghui Chen 2, Jing Lu 1, Xia Yang 3, and
Jun Yang 1,2 *
1
Zhejiang California International Nanosystems Institute, Hangzhou,
China
2
3
Sage Bionetworks, Seattle, WA, USA
Alternative splicing is an important way to regulate gene
expression. Recent studies have shown DNA damage can
induce alternative splicing. However, up to now, no systematic
study had been conducted to analyze the alternatively spliced
genes in DNA-damaged cells. Therefore, in the present study,
using GeneChipw Exon Array, we analyzed the alternatively
spliced genes in response to DNA damage induced by genotoxin benzo(a)pyrene (BaP) in HeLa cells. It was found that at
5 h after 0.5 mM BaP treatment, there were 1754 genes
showed changes in alternative splicing pattern (t-test P ,0.05,
with splicing index (SI),20.5 or SI.0.5). A more strict
analysis found 70 genes having changed splicing patterns
(t-test P , 0.01, with SI,21 or SI.1). Six genes were then
chosen for verification using RT-PCR, and the results showed
that alternative splicing did occur for genes pol n (DNA polymerase N) and CDC7 (cell division cycle 7). Taken together,
these data suggest that DNA damage indeed can induce extensive alternative splicing, and the function of those alternatively
spliced genes induced by DNA damage needs further investigation.[ This work was supported by grants from the National
Natural Science Foundation of China (Nos. 30771826,
30872140); Ministry of Science and Technology, China (No.
2009DFB30390); Department of Science and Technology,
Zhejiang Province (No. 2009C11122).]
Po14. Effect of benzo(a)pyrene on platelet
aggregation and expression of P-selectin
Qian Tang 1, Yihua Wu 2, Jing Lu 1, Tie’er Gan 2, Hu Hu 3,
and Jun Yang 1,2*
1
China
2
3
Department of Pathology and Pathophysiology, Zhejiang University
School of Medicine, Hangzhou, China
Benzo(a)pyrene (BaP) is widely presented in air pollution, coal tar and also can be found in cigarette smoke
2
China
Po13. GeneChip Exon Array reveals
extensive alternative splicing in
benzo(a)pyrene-treated HeLa cells
Abstracts
Po15. Brain toxicity of single-walled
carbon nanotubes on mice
Qiaohui Wei 1, Jun Zhang 1, Xiangjue Sun 1, Yuying Xu 1,
Guli Yang 1, Xiaomin Gu 1, Yin Zhang 1, Yifan Zheng 1,
Jun Yang 1,2, and Xinqiang Zhu 1*
1
2
China
Recent studies have indicated that nanoparticles can go
through blood brain barrier (BBB) or olfactory, enter into
the brain and cause brain damage. Single-walled carbon
nanotubes (SWCNTs) have wide applications in many
areas. However, there are very limited data about their
brain toxicity. In this study, the effects of SWCNTs on the
central nervous system in mice through tail intravenous
injection were evaluated. Using Nissl staining, it was found
that cellular morphology changed and the number of Nissl
bodies decreased in cortex after short-term exposure, but
no obvious alteration was found in corpus striatum and hippocampus. Western blot analysis indicated that glial fibrillary acidic protein (GFAP) level increased in hippocampus
and cortex after short-time exposure, but gradually
decreased to normal level after longer exposure. The neuronal nitric oxide synthase (nNOS) content was slightly
decreased only in cortex, while the heme oxygenase
(HO-1) content had no obvious changes in this study.
Taken together, this research shows SWCNTs can affect
central nerve system and cause brain damage. However,
further experiments are needed to elucidate the mechanism
of neurotoxicity and BBB damage by SWCNTs. [This
Po16. Effects of genotoxic agents on
contractile function of thoracic aorta in
Sprague-Dawley rats
Tie’er Gan 1, Yihua Wu 1, Jing Lu 2, Suping Xiao 1, Hu Hu 3,
and Jun Yang 1,2*
1
2
China
3
Genotoxins have been reported to be associated with cardiovascular complications. However, the detailed mechanism of genotoxin effects on cardiovascular system is not
clear. We thus evaluated the possible vascular effects of
benzo(a)pyrene (BaP) (an indirect-acting genotoxin) and
cisplatin (a direct-acting genetoxin), specifically on contractile function of thoracic aortic rings dissected from
Sprague-Dawley rats. Contraction of aortic ring was
induced by 60 mM kalium chloratum (KCl) and 1026 M
phenylephrine (PE) in an ex vivo perfusion system. It was
found that during 6 h incubation, cisplatin (200 mM) counteracted KCl- and PE-induced contraction by 57.6% and
91.8% in endothelium-intact aortic rings respectively.
Similar results were found in endothelium-denuded aortas.
In addition, cisplatin significantly decreased the viability of
A7r5 cell line in vitro and caused a severe damage to
blood vessel walls in vivo. In contrast, BaP (100 mM) did
not induce any changes in contractile function of aortic
rings during the ex vivo perfusion. However, after four
weeks of BaP injection intraperitoneally (10 mg/kg, once a
week) in SD rats, the maximum aortic contractile responses
to PE, KCl and acetylcholine (ACh) was significantly
and grilled food. As a carcinogen, its mode of action and
the ability to induce DNA damage have been well studied.
However, few studies have been conducted to investigate
its effect on platelet activation. Therefore, in the present
study, the effects of BaP on platelet aggregation and
expression of P-selectin were examined. It was found that
BaP itself did not induce platelet aggregation. On the other
hand, while BaP preincubation failed to enhance platelet
aggregation under collagen and thrombin stimulation, BaP
preincubation significantly enhanced ADP-induced platelet
aggregation. Using whole blood flow cytometry, the
expression of P-selectin was also evaluated. The results
showed that BaP preincubation also increased
ADP-induced
P-selectin
expression
but
not
thrombin-induced P-selectin expression. These data indicate that BaP can stimulate ADP-induced platelet aggregation and P-selectin expression, probably through the
interaction with ADP-mediated signal pathway. [This work
was supported by grants from the National Natural Science
(No. 2009C11122).]
Abstracts
decreased by 25.0%, 34.2% and 10.4% respectively, compared with vehicle-treated rats. These results suggest, in
accordance with their genotoxic properties, the cardiovascular toxicity of cisplatin is mainly due to the direct cytotoxicity, while metabolic activation is a prerequisite for
BaP-induced cardiovascular toxicity. In conclusion, genotoxins may influence the cardiovascular system in different
ways depending on their action modes. [This work was
supported by grants from the National Natural Science
(No. 2009C11122).]
cisplatin-induced DNA damage. [This work was supported
by grants from the National Natural Science Foundation of
China (Nos. 30771826, 30872140); Ministry of Science and
Technology, China (No. 2009DFB30390); Department of
Science and Technology, Zhejiang Province (No.
2009C11122).]
Po18. Benzo(a)pyrene augments platelet
activation and arterial thrombosis via
up-regulation of P38MAPK
Yihua Wu 1, Qian Tang 2, Jing Lu 2, Tie’er Gan 1, Hu Hu 3,
and Jun Yang 1,2*
Wei Wu 1,2, Chunlan Yan 1, Tie’er Gan 1, Zhanghui Chen 1,
Xianghong Lu 3, Penelope J. Duerksen-Hughes 4,
Xinqiang Zhu 1, and Jun Yang 1 *
1
2
Institute of Hygiene, Zhejiang Academy of Medical Sciences,
Hangzhou, China
3
Department of Pharmacy, Lishui People’s Hospital, Lishui, China
4
Department of Basic Sciences, Division of Biochemistry, Loma
Linda University School of Medicine, Loma Linda, CA, USA
Cisplatin has been widely accepted as one of the most
efficient anticancer drugs for decades. However, the mechanisms for the cytotoxic effects of cisplatin are still not fully
understood. Cisplatin primarily targets DNA, resulting in
the formation of DNA double strand breaks and eventually
causing cell death. In this study, we applied twodimensional electrophoresis coupled with LC-MS/MS to
analyze the nuclear proteome of HeLa cells treated with cisplatin, in an effort to uncover new mechanistic clues regarding the cellular response to cisplatin. A total of 19 proteins
were successfully identified, and these proteins are involved
in a variety of basal metabolic and biological processes in
cells, including biosynthesis, cell cycle, glycolysis and
apoptosis. Six were related to the regulation of mRNA splicing, and we therefore asked whether the Fas gene might
undergo alternative splicing following cisplatin treatment.
This proved to be the case, as the splicing forms of Fas were
modified in cisplatin-treated HeLa cells. This work provides
novel information, from the perspective of the nuclear
response, for understanding the cytotoxicity caused by
1
Health, Hangzho, China
2
China
3
Benzo(a)pyrene (BaP), a major environmental pollutant
and component of cigarette smoke, is both carcinogenic
and atherogenic in experimental models. As platelet plays
critical roles in atherothrombogenesis, we studied the effect
of BaP on platelet activation and its underlying mechanisms. BaP dose-dependently enhances platelet aggregation
induced by ADP. It augmented ADP-induced platelet
P-selectin expression and fibrinogen binding. Under an
arterial shear rate of 1000 s21, BaP increased platelet
adhesion on collagen surfaces by 20%. Western blot analysis showed that BaP could increase P38MAPK phosphorylation in ADP-induced platelet aggregation model,
indicating the possible involvement of this signaling
pathway. These in vitro results were further examined in
FeCl3-induced arterial thrombosis mouse model. The
results showed that BaP increased the carotid arterial occlusion time by 25% compared with wild-type mice (P ,
0.01). BaP also augmented ADP-induced platelet activation
via the up-regulation of P38MAPK pathway, and enhanced
arterial thrombus formation in vivo. Taken together, our
data suggest that BaP may contribute to atherothrombogenesis, probably via the platelet signaling pathways. [This
Science Foundation of China (Nos. 30771826, 30872140);
Po17. Nuclear proteome analysis of
cisplatin-treated HeLa cells
Abstracts
Po19. Cytotoxic and genotoxic effects of
multi-wall carbon nanotubes on human
umbilical vein endothelial cells in vitro
Po20. Effects of multi-walled carbon
nanotubes on the early stage of
atherosclerosis in rats
Yuanyuan Guo 1, Yifan Zheng 1, Jun Zhang 1, Jun Yang 1,2,
and Xinqiang Zhu 1 *
Yuying Xu 1, Yuanyuan Guo 1, Jie Yang 1, Guili Yang 1,
Qiaohui Wei 1, Yifan Zheng 1, Jun Yang 1,2, and
Xinqiang Zhu 1 *
1
2
China
2
China
The biomedical application of multi-walled carbon nanotubes (MWCNTs) requires a clear understanding of their
toxicity after intravenous administration. However, little is
known about their effects on atherosclerosis or endothelial
cells. In this study, the short-term effects of intravenously
injected MWCNTs at the early stage of atherosclerosis in
rats were studied. Serum biochemical parameters (HDL,
CHOL, and TG) were measured. Histological observations
demonstrated that severe inflammation and inflammatory
cell infiltration occurred in lung, while fat vacuoles
reduced in liver, and vascular calcification exacerbated in
aorta. Furthermore, it was shown that MWCNTs can cause
damage in endothelial cells, change the endothelial permeability and induce cells to secrete cytokines such as
VWF and sICAM-1. Taken together, these data suggest
that MWCNTs may promote the early stage of atherosclerosis formation through inflammation. [This work was
Foundation of China (No. 30771824, 30800923); Ministry
(No. 2009C11122).]
Po21. Effects of alkylating agent methyl
methanesulfonate (MMS) on
cardiovascular system
Tie’er Gan 1, Suping Xiao 1, Yihua Wu 1, Jing Lu 2, Hu Hu 3,
and Jun Yang 1,2*
1
2
China
3
The genotoxin methyl methanesulfonate (MMS) is an
alkylating agent that predominantly methylates nitrogen
Carbon nanomaterials have multiple applications in
various areas. However, it has been suggested that
exposure to nanoparticles may be a risk for the development of vascular diseases due to injury and dysfunction of
the vascular endothelium. Therefore, in the present study,
the cytotoxic and genotoxic effects of multi-wall carbon
nanotubes (MWCNTs) on human umbilical vein endothelial cells (HUVECs) were evaluated. Optical and transmission electronic microscopy (TEM) study showed that
MWCNTs were able to enter cells rapidly, distribute in the
cytoplasm and intracellular vesicles and induce morphological changes. Exposure to MWCNTs reduced the viability of HUVECs, and induced apoptosis in HUVECs.
Furthermore, MWCNTs could cause DNA damage as indicated by the formation of gH2AX foci. MWCNTs also
affected cellular redox status, e.g., increasing intracellular
reactive oxygen species (ROS) and malondialdehyde
(MDA) levels, as well as altering superoxide dismutase
(SOD) activity and glutathione peroxidase (GSH-Px)
levels. On the other hand, the free radical scavenger
N-acetyl-L-cysteine (NAC) preincubation can inhibit the
cytotoxic and genotoxic effects of MWCNTs. Finally,
MWCNTs did not induce the generation of tumor necrosis
factor-a (TNF-a), indicating no significant inflammation
response was evoked. Taken together, these results demonstrated that MWCNTs could induce cytotoxic and genotoxic effects in HUVECs, probably through oxidative
damage pathways. [This work was supported by grants
from the National Natural Science Foundation of China;
Zhejiang Province (No. 2009C11122). ]
1
Abstracts
Po22. Effects of multiwall carbon
nano-onions on platelet aggregation and
coagulation system
Guili Yang 1, Yuying Xu 1, Qiaohui Wei 1, Yifan Zheng 1,
1
2
China
Recent studies have suggested that nanomaterials may
have various effects on the blood system. In the present
study we evaluated the effects of multiwall carbon
nano-onions (MWCNOs) on platelet aggregation and blood
coagulation. It was found that in vitro, MWCNOs exhibited
potent and broadspectrum inhibitory effects on rat platelet
aggregation caused by various stimulators in a
concentration-dependent manner, with a highest inhibition
rate of 59%. In vivo study revealed that MWCNOs inhibited rat platelet aggregation induced by ADP as well, but
did not affect activated partial thromboplastin time
(APTT), prothrombin time (PT), thrombin time (TT),
bleeding time (BT) and platelet count (PC). These findings
indicated that the effects of the MWCNOs on the blood
system might be mediated by its inhibition on platelet
aggregation rather than coagulation system, and that further
mechanistic study for the inhibition effect of MWCNOs on
platelet aggregation is needed. [This work was supported
by grants from the National Natural Science Foundation of
China (No. 30771824, 30800923); Ministry of Science and
Technology, China (No. 2009DFB30390); Department of
Science and Technology, Zhejiang Province (No.
2009C11122).]
Po23. Single-walled carbon nanotubes
induces oxidatve stress in mice brain
Qiaohui Wei 1, Jun Zhang 1, Xiaomin Gu 1, Yuying Xu 1,
Guli Yang 1, Xiangjue Sun 1, Yin Zhang 1, Yifan Zheng 1,
1
2
China
Broad biomedical applications of single-walled carbon
nanotubes (SWCNTs) have raised concerns on their biosafety effect. Accumulating evidence has shown that nanoparticles can enter the brain and cause brain damage. In
this research, we studied the short-term oxidative stress
effects of SWCNTs on four different brain sections in
mice, namely cerebellum, corpus striatum, hippocampus
and cortex, through tail intravenous injection. Oxidative
damage related factors including superoxide dismutase
(SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) content were measured in these tissues. It
was found that SWCNTs exposure caused reduced antioxidase activity and elevated lipid peroxidation levels in
corpus striatum, hippocampus and cortex, but not cerebellum in a dose-dependent manner. Oxidative damage was
observed in corpus striatum. These data indicate that
SWCNTs could affect the antioxidant system, and cause
oxidative damage in mice brain. Nevertheless, further
experiments are needed to elucidate specific mechanism
and the long-term effects of SWCNTs on the brain. [This
atoms on purines. Its genetic toxicity and underlying mechanism for DNA damage have been studied extensively.
However, its effect on cardiovascular system has not been
evaluated, even though evidence has been accumulating
pointing to the possible adverse cardiovascular effects elicited by genotoxic agents. Therefore, in the present study,
we examined the effects of MMS on cardiovascular system
in Sprague-Dawley (SD) rats by measuring its effects on
peripheral blood cells, the structure of vascular wall, thoracic aorta tension and blood pressure. It was found that 1
day after MMS (low: 25 mg/kg; high: 60 mg/kg) injection
intraperitoneally in SD rats, white blood cells were significantly destructed by both doses, while the red blood cells
and platelets were not affected. In addition, the structure of
vascular wall, vascular tension and blood pressure were not
affected in MMS-treated rats compared with vehicle ones.
Similar results were found in SD rats 3 days after MMS
treatment. These results suggest that the cytotoxicity of
MMS may be responsible for the decrease in white blood
cells. Taken together, acute treatment of MMS has no significant effect on the cardiovascular system, which may be
partly due to its rapid metabolism in vivo. [This work was
(No. 2009C11122)]
Abstracts
Po24. Preparation and biological activity
of aqueous nanoparticle suspension of
fullero-valine
Feng Kang, Yanxia Wang, and Gaoguang Song
Po25. Effects of iasmine, jasmine tea
on immune activity
Mi Wang 1 , Junliang Jiang 1, Xiaocong Kuang 2, Jing Qin 3,
Bingning Ou 3, and Haidong Li 3
1
School of Life Science, Beijing Institute of Technology, Beijing, China
To observe the immune effects of the jasmine tea extract
and the extract components of jasmine, leaching solution of
jasmine tea was prepared with the water formulation.
Jasmine de-cerebrovascular oil was prepared using petroleum ether by parallel column chromatography. The
immunomodulatory function of jasmine tea was evaluated
by the spleen lymphocyte transformation test and the neutrophil phagocytosis assay. The effects of jasmine
de-cerebrovascular oil and B-II component on lymphocyte
proliferation were measured by MTT assay. Leaching solution of jasmine tea can significantly improve the rate of
lymphocyte transformation in mice, but had no effect on
neutrophil phagocytosis. MTT assay confirmed that
de-cerebrovascular jasmine oil and B-II can promote the
growth of mouse lymphocytes. This study showed that
leaching solution of jasmine tea, jasmine de-cerebrovascular
oil and B-II had certain effects on the immune system. In
particular, leaching solution of jasmine tea can increase the
lymphocyte transformation rate. The results provide new
information regarding the immunomodulatory effects of
extracts from traditional Chinese medicine.
Po26. Expression and purification of
mutant trichosanthin in Escherichia coli
Chengcheng You, Weihong Cao, Liming Huang,
Yiling Huang, and Zhaoxiang Zhang
Department of Pathology, Medical school of Three Gorges
University, Yichang, China
To construct a mutated trichosanthin (mTCS), four
amino acids (aa 120-123) are deleted. To express and
purify this mTCS in E. coli, cDNA encoding mTCS was
obtained by PCR-driven overlap extension, and then
cloned into pET-28a(þ) vector. After restriction and
sequence
analysis,
the
recombinant
plasmid
pET-28a(þ)-mTCS was transformed into BL21 (DE3).
The mTCS was expressed by IPTG induction and purified
by Ni-NTA resin. A mutated TCS was obtained and a protocol for prokaryotic expression and purification of this
mTCS was established successfully.
Aqueous nanoparticle suspensions of fullerene and its
derivatives are currently attracting much attention. In this
study, a novel fullero-valine (Fval) with free a-amino
group was synthesized by three chemical reactions and
characterized by different spectra of MALDI-TOF-MS,
UV-Vis, FT-IR and 1H-NMR. First, N-substituted 3,4-fulleropyrrolidine was synthesized and its characterization
results were consistent with its structure. Then,
Boc-fullero-valine was synthesized, and finally target compound (fullero-valine) was synthesized. Characterization
results demonstrated that a pure fullero-valine was
achieved.
Fullero-valine aqueous nanoparticle suspension (n-Fval)
was the prepared and characterized by transmission electron
microscopy and Zeta-potential. The results showed that the
nanoparticles were spherical with diameters less than
200 nm. The average Zeta-potential was 2(12.0 + 0.8)
mV.
Finally, with regard to the bioactivity, the nanoparticle suspension could inhibit the polymerase chain reaction (PCR)
and Taq DNA polymerase activity. With dose of n-Fval at
60 mM the PCR was completely inhibited. Based on the
dose-response curve, half inhibition concentration (IC50) of
n-Fval was calculated as 10 mM. In a PCR with 60 mM
n-Fval, PCR can be resumed gradually with increased
amount of enzyme. The inhibition of n-Fval was reversed by
80 % with an enzyme amount of 40 U. The singlet oxygen
inhibitor sodium azide and hydroxyl radical scavenger mannitol had no effects on enzyme itself at the concentrations of
2, 6 and 10 mM, and could not reverse the inhibition of
n-Fval on PCR, indicating reactive oxygen species may not
be involved in the inhibition effects of n-Fval. Taken
together, the data suggested that fullero-valine and its
aqueous nanoparticle suspensions may have the potential
application in biomedicine as a novel enzyme inhibitor.
Health Management Institute of Guangxi, Nanning, China
Department of Pathophysiology, Guangxi Medical University,
Nanning, China
3
Department of Chemistry, Guangxi Medical University, Nanning, China
2
Abstracts
Po27. p38-targeted inhibition of
interleukin-12 expression by ethanol
extract from Cordyceps bassiana in
lipopolysaccharide-activated macrophages
Se Eun Byeon 1, Tao Yu 1, Jaehwi Lee 2 *, Gi Ho Sung 3,
Tae Woong Kim 4, Hyoung Jin Park 5, and Jae Youl Cho 1*
1
Cordyceps species have been known as traditionally valuable mushroom in Korea, China and Japan. This plant has
been found to display a lot of ethnopharmacological activities such as anti-oxidative, anti-cancer, anti-inflammatory,
anti-diabetic, and anti-obesity effects. Previously, we found
that ethanol extract of Cordyceps bassiana was able to suppress the production of IL-12 and IFN-g in macrophages
and T lymphocytes. In this study, we further explored its
molecular inhibitory mechanism using butanol fraction of
this plant (Cb-BF). Similarly, this fraction also blocked the
proliferation of T cells and the production of nitric oxide
(NO), IL-12 and IFN-g but not IL-4. Interestingly, Cb-BF
suppressed luciferase activity mediated by NF-kB, AP-1,
and STAT-1. In agreement with this, this fraction diminished
the translocation of these transcription factors into nuclear
fraction. Moreover, upstream signaling events for the activation of these factors such as Syk, JAK-2 and ERK were
clearly suppressed. Therefore, these results suggest that
Cordyceps bassiana may be a multifunctional ethnomedicinal plant with anti-inflammatory principles.
Po28. Water extract of Sorbus commixta
with Src and Syk inhibitory activities
inhibits macrophage-mediated
inflammatory responses
Tao Yu, Yong Gyu Lee, Se Eun Byeon, Ting Shen,
Min Hyo Kim, Dae Sung Yoo, and Jae Youl Cho *
School of Bioscience and Biotechnology, and Institute of Bioscience
and Biotechnology, Kangwon National University, Chuncheon, Korea
Sorbus commixta has been known as an enthopharmacologically valuable plant in Korea, China and Japan. This
Po29. Carbon black nanoparticles induce
oxidative stress in A549 cells
Min Yu 1 , Riping Chen 2, Dongmiao Cai 2, Zhenyu Jia 1, and
Xing Zhang 1
1
Labor Health & Occupational Disease Lab, Institute of Hygiene,
Zhejiang Academy of Medical Sciences, Hangzhou, China
2
Electronic Microscope & Pathologic Morphology Lab, Institute of
Hygiene, Zhejiang Academy of Medical Sciences, Hangzhou, China
The present study aimed to examine the oxidative stress
caused by exposure to carbon black nanoparticles in A549
cells. Carbon black nanoparticles used in this study is
carbon pearl 480 ( produced by Cabot Corporation, with an
average size of 25-60 nm). Both MTT assay and Typan
blue assay were applied to determine the non-toxic concentration of the nanoparticles on A549 cells.
Twenty-four-hour exposure of the nanoparticles at different
concentrations, namely 0, 25, 50 and 100 mg/ml, did not
School of Bioscience and Biotechnology, and Institute of Bioscience
and Biotechnology, Kangwon National University, Chuncheon, Korea
2
College of Pharmacy, Chung-Ang University, Seoul, Korea
3
Department of Botany and Plant Pathology, Oregon State University,
Corvallis, OR, USA
4
Department of Biochemistry, Kangwon National University,
Chuncheon, Korea
5
Department of Physiology, College of Medicine, Hallym University,
Chuncheon, Korea
plant has been known to exhibit numerous pharmacological
activities such as anti-oxidative, anti-cancer, anti-lipid peroxidation, anti-atherogenic, and vasorelaxant effects. A
number of pharmacological potentials have been demonstrated, but anti-inflammatory effect of this plant and the
molecular mechanisms have not been fully investigated
yet. To address its anti-inflammatory activity and inhibitory
mechanism, lipopolysaccharide (LPS)-stimulated macrophages were chosen and their production of inflammatory
mediators was evaluated. Water extract (Sc-WE) from
S. commixta strongly suppressed the production of nitric
oxide (NO) and prostaglandin (PG)E2 in a dose-dependent
manner. The extract also clearly diminished the mRNA
levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, indicating that the inhibition occurs at transcriptional level. Interestingly, Sc-WE remarkably blocked
NF-kB translocation and its upstream signaling events consisted with inhibitor of kBa (IkBa), IkBa kinase (IKK),
Akt (protein kinase B), phosphoinositide-dependent kinase
1 (PDK1), and p85/phosphoinositide-3-kinase (PI3K),
according to a reporter gene assay and an immunoblotting
analysis. More intriguingly, Sc-WE strongly suppressed
kinase activities of Src and Syk as well as their phosphorylation levels without altering molecular complex formation between them and toll like receptor (TLR)-4 or
MyD88, an adaptor protein of TLR4-mediated signaling.
Therefore, our results suggest that Sc-WE may have strong
anti-inflammatory activity by suppressing an inflammatory
signaling cascade composed of Src, Syk and NF-kB.
Further in vivo efficacy study will be followed to evaluate
its therapeutic application.
Abstracts
decrease cell viability. However, 200 or 400 mg/ml of the
nanoparticles significantly reduced cell viability. To
demonstrate the nanoparticles-induced oxidative stress,
reactive oxygen species (ROS), superoxide dismutase 2
(SOD2) and lipid peroxidation were measured. Exposure of
the nanoparticles (0, 25, 50 and 100 mg/ml) to A549 for
6 h or 24 h elevated the ROS level in a dose-dependent
manner, decreased the expression of SOD2 and increased
TBARS. In summary, carbon black nanoparticles resulted
in increased oxidative stress without obvious cell death.
Po30. The role of alcohol consumption
in cancer development in the Chinese
population: a systematic review and
meta-analyses
conclusion, health programs focused on helping patients
limit alcohol intake may be important for the prevention of
major cancers in China. Further studies are needed to
examine the environmental interaction with alcohol in the
development of various cancers in Chinese and other
populations.
Po31. MC-LR-induced PP2A inhibition is
associated with alterations of expression
levels and post-translational modifications
of PP2A-C subunit in PC12 cells
Guanmin Meng and Lihong Xu
Department of Biochemistry and Genetics, School of Medicine,
1
Department of Social Medicine and Health Service Management,
College of Preventive Medicine, Third Military Medical University,
Chongqing, China
2
Department of Hygienic Toxicology, College of Preventive
Medicine, Key Lab of Medical Protection for Electromagnetic
Radiation, Ministry of Education of China, Third Military Medical
University, Chongqing, China
*Correspondence address. Tel: þ86-23-68752271;
Alcohol consumption is increasing rapidly worldwide
and is associated with numerous cancers. This study examined the role of alcohol in the incidence of cancer development in Chinese population. 129 case-control studies and 5
cohort studies on the association between alcohol consumption and 13 types of cancer in China were identified
for meta-analysis by searching Medline/PubMed,
EMBASE, CNKI and Chinese Journal of Science and
Technology of VIP. Meta-analyses of cohort studies found
that alcohol consumption was a risk factor for gastric
cancer [ pooled RR (95%CI): 1.14 (1.02, 1.28)], but its role
in esophageal cancers (EC) and lung cancer occurrence
was not significant (P . 0.05). Meta-analyses of casecontrol studies identified alcohol consumption as a risk
factor
for
EC,
gastric
cancer,
hepatocellular
carcinoma(HCC), colorectal cancer, pancreatic cancer,
nasopharyngeal cancer and oral cancer [ pooled OR
(95%CI): 1.84 (1.46, 2.32), 1.36 (1.22, 1.53), 1.52 (1.25,
1.87), 1.54 (1.06, 2.23), 1.15 (1.01, 1.32), 1.21 (1.05,
1.40) and 1.71(1.30, 2.24) respectively]; but it was identified as a protective factor for breast cancer and gallbladder
cancer [ pooled OR (95%CI): 0.76 (0.64, 0.92) and 0.71
(0.53, 0.96) respectively]; its role in lung cancer, prostate
cancer, ampulla of vater cancer, and extrahepatic cholangiocarcinoma was not significant (P . 0.05). In
Microcystin-LR, a potent inhibitor of protein phosphatase 1 and 2A, is one of the cyclic peptide toxins implicated in several livestock and human diseases. PP2A
heterotrimer is the major functional form in vivo, composed
of a structural A subunit, a regulatory B subunit and a catalytic C subunit. Besides PP2A inhibitors, activity of PP2A
can be determined by holoenzyme composition and posttranslational modifications of the catalytic subunit including phosphorylation and methylation. Although the toxicity
of microcystins has been recognized due to the specific
inhibition of PP2A, the underlying molecular mechanism
remains to be answered.
The present study was undertaken with the objective of
evaluating (i) protein phosphatase 2A activity, (ii) the
protein profile of PP2A composition subunits including A
subunit, C subunit, as well as the regulatory B family subunits, Bband Bg, and (iii) methylation and phosphorylation
of PP2A-C as well as protein phosphatase methylesterase-1
(PME-1) expression after 6h MC-LR exposure in PC12
cells.
Six hours after MC-LR exposure, a strong dosedependent inhibition of PP2A activity was observed, as
well as decreased C subunit expression and compensatory
enhanced A subunit level. It is well known that phosphorylation at Tyr307 of PP2A-C lead to its decreased
phosphatase activity. In the present study, concentrationdependent increase of phosphorylated PP2A-C at Tyr307
was consistent with that of PP2A inhibition, which
suggested that MC-LR inhibited PP2A activity not only by
down-regulation of C subunit, but also through the
up-regulation of phosphorylated PP2Ac. It is reported that
PME-1 inhibits PP2A activity by demethylation of the
PP2A-C at Leu309, and changes in methylation of
PP2A-C could also modulate holoenzyme assembly. In the
Ying Li 1, Huan Yang 2, and Jia Cao 2 *
Abstracts
Po32. Effect of microcystin-LR on PP2A
in human amniotic epithelial cells
Jing Liang and Lihong Xu
The increased exposure of microcystins (MCs) has
become a global issue. MCLR, which is one of the MCs
congener, can inhibit the activity of Protein Phosphatase
2A (PP2A) through binding to the catalytic subunit. PP2A
is the major serine/threonine phosphatase in eukaryotic
cells, consisting of a structural A subunit, a catalytic C and
a regulatory B-type subunit. To further dissect the role of
PP2A in MCLR-induced toxicity, the present study was
undertaken. After exposure of FL cells to MCLR for 6 h,
experiments including real-time PCR, western blot analysis, phosphatase activity analysis, immunofluorescence and
cell cycle analysis were performed. PP2A activity as well
as the expression of C subunit was remarkably increased
by exposure to 500 nM MCLR. The phosphorylation of
PP2Ac at Tyr307 was decreased after treatment with
MCLR. Furthermore, exposure to MCLR for 6 h induced
enhanced C subunit of PP2A staining in nucleus.
Treatment with MCLR induced a small increase in the G1/
S cell population. These results revealed for the first time
that exposure to MCLR involves up-regulation of PP2A
phosphatase activity plus increased expression of C
subunit, which may lead to further investigation on the
mechanism of toxicity induced by MCLR. [Grants: the
National Nature Science Foundation of China (30771827,
20777067); the Key Special Program on the S&T of China
for the Pollution Control and Treatment of Water Bodies
(2008ZX07421-001)]
Po33. MAPK pathways and microtubule
depolymerization are involved in
tributyltin-induced apoptosis in human
amnion cells
Yali Zhang and Lihong Xu
Department of Biochemistry and Genetics, School of Medical,
Tributyltin (TBT), a highly toxic environmental contaminant, has been shown to induce mitochondrial-dependent
apoptosis in several mammalian cells. However, the initial
signal transduction pathways involved in TBT-induced
apoptosis are still not fully elucidated. In the present study,
the serine/threonine protein phosphatase (PP)2A, mitogenactivated protein kinases (MAPKs) cascade pathway and
microtubule/tubulin were investigated in TBT-induced
apoptosis in human amnion cells for the first time.
Apoptosis was determined by flow cytometry. Expression
of phosphorylated ERK1/2, JNK and p38 were assessed by
western blot. The effect of TBT on tubulin was detected
with immunofluorescence confocal microscopy. TBT effectively induced apoptosis in FL cells in a dose-dependence
manner. JNK and p38 as well as the downstream transcription factor c-jun and ATF were activated, while ERK cascades that involves upstream activator Raf, MEK1/2 and
downstream effector, c-myc were strongly inhibited.
Furthermore, the increase in the phosphorylation of JNK
and p38 was accompanied by an inhibition in pp2A activities. In addition, confocal microscopic analysis revealed
reduction of microtubules density and the disappearance of
the tubulin filaments in the cytoplasm occurring 1 h after
TBT treatment. Finally, we also examinationed whether
TBT had the effect on cell cycle-related signal pathway
and cell cycle progress. Results from the present study
showed that the expression of the cell cycle phosphatase
Cdc25C was decreased and the consequent phosphorylation of Cdc2 at Tyr15 site was upregulated in response to
1-6 mM TBT treatment for 1 h, however, cell cycle progression was not affected. Taken together, our results
suggest that inhibition in PP2A activity, activation of JNK/
p38 MAPK signal cascades and inactivation in ERK signal
cascades, as well as depolymeration of tubulin might be
involved in TBT-induced apoptosis events. [Grants: the
present study, increased PME-1 level was concurrent with
decreased PP2A-C methylation, strongly implying that
MC-LR induced PME-1-mediated PP2A demethylation,
which might be involved in changes of PP2A heteromultimeric composition and PP2A activity inhibition.
Furthermore, the present study demonstrated that treatment
of MC-LR increased protein levels of Bg while Bb was
not affected, indicating that that MC-LR induced changes
in holoenzyme assembly.
Taken together, PP2A inhibition induced by MC-LR is
the result of the combined effects of alterations of various
composition subunits levels that indicated changes in
holoenzyme assembly, PP2A-C Tyr307 hyperphosphorylation and PME-1 mediated PP2A-C Leu309 demethylation.
This study provides a new insight into the effect of
MC-LR on PP2A. [Grants: the National Nature Science
Foundation of China (30771827, 20777067); the Key
Special Program on the S&T of China for the Pollution
Control
and
Treatment
of
Water
Bodies
(2008ZX07421-001)]
Abstracts
National Nature Science Foundation of China (30771827,
20777067); the Key Special Program on the S&T of China
for the Pollution Control and Treatment of Water Bodies
(2008ZX07421-001)]
Po34. Effect of MCLR on PP2A in
Hek293 cells
Tan Li and Lihong Xu
Session 2: Systems Biology and
Biomarkers
Po35. MicroRNAs as a potential agent to
DNA damage response in human liver
cancer cell
Gaofeng Liang, Youhua Shao, Yanliang Zhu, and
Zhongdang Xiao *
State Key Laboratory of Bioelectronics, School of Biological Science &
Medical Engineering, Southeast University, Nanjing, China
*Correspondence address. þ86-25-83790820; E-mail: zdxiao@seu.
edu.cn
Microcystin-LR (MCLR) is a cyanobacteria-produced
cyclic heptapeptide toxin which exerts potent inhibition of
protein phosphatases (PP) including PP1, PP2A, PP2B,
PP4 and PP6 in cells. Thus, MCLR is considered as a
tumor promoter, probably by causing hyperphosphorylated
state in cells and thereby perturbing cell signal transduction. However, the precise mechanism affected by MCLR
has not been fully elucidated. PP2A, a heterotrimeric
holoenzyme, is a major protein phosphatase in cell, which
is regarded as a tumor suppressor. To investigate how
MCLR affects PP2A activity as well as its subunits
expression, Hek293 cells were treated by MCLR from relatively low dose (100 nM) to relatively high dose
(10,000 nM) for 24 h. MCLR uptake was confirmed by
immunofluorescence and western blot using a specific antibody. PP2A activity was measured with a Promega kit and
the protein level of PP2A subunits were detected by
western blot. Morphological changes were visualized by
immunofluorescent microscopy. MCLR was accumulated
in cells and specifically bound to the catalytic subunit of
PP2A and PP1. PP2A activity underwent a statistically significant increase to approximately 130% at low dose
(2000 nM), followed by a significant decrease to about
20% at high dose (10,000 nM), which correlated with the
PP2A catalytic subunit expression. Morphological changes
with cellular volume shrinkage at high dose treatment were
observed. This study demonstrated the toxicity mechanism
of MCLR from the angle of PP2A. Our results suggested
that PP2A is a target of MCLR, which may convey a wide
range of physiological changes in cells. This study may
help further researches on the MCLR toxicity as well as
specific function of PP2A. [Grants: the National Nature
Science Foundation of China (30771827, 20777067); the
Key Special Program on the S&T of China for the
Pollution Control and Treatment of Water Bodies
(2008ZX07421-001)]
MicroRNAs (miRNAs) are important effector molecules
of RNA interference (RNAi). They regulate gene
expression at post-transcriptional level and thereby control
cellular mechanisms including development, differentiation, proliferation, apoptosis and organ morphology.
miRNAs are generally down-regulated in various cancer
types; although they have been found aberrantly overexpressed [1,2]. miRNAs expression is also affected by cellular stress, such as radiation and chemotherapy. In this
research, we monitored the expression levels of several
miRNAs by employing the stem-loop real-time polymerase
chain reaction(PCR) in HepG2 cells treated with
UV-radiation [3].
The human liver cancer cell lines (HepG2) were cultured
in 25 cm2 flask in the medium of DMEM supplemented
with 10% fetal bovine serum and 1% Penicillin–
Streptomycin with 5% CO2 at 378C. The cells were randomly divided into 4 groups which were exposed to 0, 50,
70 and 100 J m2 of UVB separately. After irradiation, the
cells were cultured in DMEM again for 0, 2, 4, and 12 h.
MTT assay was determined Cell viability, and total RNA
was extracted by using Trizol reagent from HepG2 cells
irradiated with 50 J m2 UVB (2, 4, and 12 h postirradiation) and the control group. The miRNA sequence-specific
primers for miR-21, miR-26a, miR-34a, miR-221,
miR-181a and endogenous control U6 RNA were selected
for monitor the expression of the miRNAs.
Among the selected miRNAs, we observed a significant
increase in the expression of tumor-suppressor miR-26a,
miR-34a. The expression changes of miR-34a and
miR-26a may be involved in important protective mechanisms counteracting radiation damage. Additionally, these
cellular changes may constitute an attempt to counteract
radiation-induced DNA damage. Bioinformatics analysis
suggests that this is partly through control of the protooncogene homologue P53 and genes in the DNA damage
Abstracts
response pathway [4]. These findings were the first direct
evidence that miRNAs could suppress resistance to anticancer cytotoxic therapy, a common feature of cancer cells.
More than half of cancer patients received radiation treatment. Although radiation treatment is very effective, it still
need develop radiation-sensitive agent to achieve tumor
specific treatment. Using liver cancer cell lines, we identified several miRNAs, which may be involved in radiation
sensitivity. One of special interest is miR-26a which is
shown to specifically sensitize liver cancer cells against
radiation treatment. Therefore, miR-26a could be a potentially good target to enhance the efficacy of current cancer
therapies.
References
Po36. Alteration of microRNA expression
in mouse hippocampal after traumatic
brain injury and hypothermia traumatic
brain injury
Qiongfang Ruan 2, Yang Wang 2, Changfu Pan 1,
Xiaoli Shen 1, Zhucai Kuang 1, Lei Wu 1, Xianliang Lai 1,
and Zhifeng Deng 1 *
1
Department of Neurosurgery, the Second Affiliated Hospital of
Nanchang University
2
Institute of Urology of Nanchang University, Nanchang China
*Correspondence address. E-mail: [email protected]
microRNAs are 22 nucleotide long, nodcoding RNAs
that control cellular function by either degrading mRNAs
or arresting their translation. miRNAs are known to regulate many improtant cellular processes, including proliferation, apoptosis, neurogenesis, and morphogenesis. Many
cellular processes were changed after traumatic brain injury
(TBI). For the past few years, extreme climate happened
frequently, and gradually increased. It has not yet been
known how miRNAs expression is altered and what the
regular function of miRNAs in the TBI and hypothermia
1 Lu J, et al. MicroRNA expression profiles classify human cancers. Nature
2005, 435: 834 – 838
2 Gaur A, et al. Characterization of microRNA expression levels and their
biological correlates in human cancer cell lines. Cancer Res 2007, 67:
2456– 2468
3 Chen CF, et al. Real-time quantification of microRNAs by stem-loop
RT-PCR. Nucleic Acids Res 2005, 33: e179
4 He L, et al. microRNAs join th p53 network-another piece in the tumoursuppression puzzlee. Nat Rev Cancer 2007, 7: 819 –822
TBI pathophysiology is liked. In the present study, the
mice were suffered closed head injury, diveded into tow
groups, one was put in the room temperature called injury
group, the other was put in 48C called hypothermia group,
then mouse hippocampal were harvested after 4 h. Total
RNA was extracted from 100 mg of tissue by using the
mirVana total RNA extraction kit (Applied Biosystems/
Ambion). The quality and quantity of the RNA was evaluated by 260/280 ratio and Agilent 2100 Bioanalyzer
(Agilent Technologies). Microarrays from Agilent
Technologies were used to examine microRNAs changes
in the hippocampal expression levels of two groups at 4 h,
compared with sham-operated group (three arrays/group).
Raw data are analyzed with GeneSpring GX software.
microRNAs concerned with angiogenesis, such as mir-92a,
mir-320, are chosen to validate by quantitative real-time
ploymerase chain reaction. Our research revealed that comparison to sham-operated group, 257 miRNAs were
expressed in injury group, in which, expression levels of
35 miRNAs were significantly altered, 15 miRNAs were
up-regulated, and 19 miRNAs were down-regulated; 226
miRNAs were expression in hypothermia group, in which
expression levels of 15 miRNAs were significantly altered,
9 miRNAs were upregulated, and 6 miRNAs were downregulated. 9 miRNAs were significantly changed between
injury group and hypothermia group. Hierarchical clustering of the miRNAs significantly altered of 9 individual
microarrays that showed 2 distinct clusters. One contained
three samples was injury group, the other contained six
samples was hypothermia group and sham-operated group.
Bioinformatic analysis of the predicted targets for miRNAs
which are changed greatly revealed an overrepresentation
of proteins involved in several biological processes, including signal transduction, transcriptional regulation, proliferation, and differentiation. Finally, we utilized qRT-PCR
methods to verify expression of mir-92a, mir-320. The
qRT-PCR results indicated good consistency with the
results of the microarray method. The data display that
many miRNAs have changed greatly after TBI. It indicates
that many biological processes involved in TBI pathophysiology may be regulated by miRNAs. Analysis of hierarchical clustering demonstrates that the expression patters
were more similar with each other between hypothermia
group and sham-operated group. It should be validated
whether hypothermia condition play a protection role in the
traumatic brain injury process. Further study should be
done to reveal the molecule mechanism of TBI pathophysiology. [This work was supported by the grant from the
National Key Technology R&D Program (Grant No.
2008BAI68B06).]
Abstracts
Po37. Alteration of circulatory platelet
microparticles and endothelial
microparticles in patients with chronic
kidney disease
Guoyuan Lu, Shuhua Zhang, Qing Qiao, Lei Shen,
Ming Li, and Deyu Xu
Nephrology Department of the First Affiliated Hospital of Soochow
University, Suzhou, China
Faliang Gao 1, Yang Wang 2, Yuanlei Lou 2,
Qiongfang Ruan 2, Wei Tu 1, Shigang Lv 1, Changfu Pan 1,
and Zhifeng Deng 1 *
1
Nanchang University, Nanchang, China
2
Institute of Urology of Nanchang University, Nanchang, China
Stroke is a major cause of mortality and morbidity in the
population worldwide. While the incidence of stroke is
decreasing, its prevalence in the world is still increasing
because of a growing elderly population. Despite neuroprotective drugs were widely used in multicenter therapeutic
stroke trials, limit benificial was proved to those patients
suffered from stroke. Recent studies have noticed that
active angiogenesis was induced in the penumbra after
stroke, which could improve the brain injury protection.
This discovery identified angiogenesi-based strategies as a
new target for the therapy of brain ischemia, and the timely
revascularization in ischemic region is generally accepted
as the key treatment for stroke right now. Although multiple angiogenic factors such as VEGF, BDNF were shown
to play critical roles in promoting angiogenesis after stroke,
however, very little is known about the complete regulation
mechanism of this process. MicroRNA (miRNAs) was a
class of small non-coding RNA species, which participate
in the regulation of various biological processes. While
studies have shown a critical function of miRNA in
ischemia-induced angiogenesis in several tissues, the
specific miRNA that regulate angiogenesis after stroke is
still unknown. Mir-210 is known as the hypoxia-induced
miRNA; evidence showed that a significant up-regulation
of MiR-210 expression was detected in endothelial cells’
response to oxygen deprivation and thus stimulated the formation of capillaries by depressing its target gene
ephrinA3 protein level. We proposed mir-210 may be a
key regulator of angiogenesis after stroke. This study was
performed to explore the potential roles of mir-210 in
ischemia-induced angiogenesis after stroke. Rat middle cerebral artery occlusion (MACO) models were produced by
obstructing MCA using a single strand nylon thread. Rats
were killed at 1, 3, 7 days after ischemia, and the impaired
cortical areas were extracted for examine. The sham group
was not operated as control. MiRNAs were isolated from
ischemic cortex and the expression change of mir-210 was
Our aim is to compare plasma platelet microparticles
(PMPs), P-selectin, endothelial microparticles (EMPs), and
von Willebrand factor (vWF) between a normal control
group and patients with chronic kidney disease (CKD) and
to explore the significance of PMPs and EMPs in CKD.
Levels of plasma PMPs, P-selectin, EMPs and vWF in 122
CKD patients and 20 normal controls were measured by
flow cytometry and enzyme-linked immunosorbent assay
(ELISA). Relationships between PMPs, EMPs and blood
pressure, creatinine clearance rate, 24 h urine protein,
hemoglobin, and cholesterol were analyzed. Plasma PMPs,
P-selectin, EMPs and vWF levels in CKD patients were
significantly higher than those of the control group. Plasma
PMPs and P-selectin levels for nephrotic syndrome (NS)
were significantly higher than for other CKD groups. No
significant difference was found between other CKD
groups. Plasma EMPs and vWF in NS, lupus nephritis
(LN) and hypertensive nephropathy groups were significantly higher than that of diabetic nephropathy (DN) and
chronic glomerulonephritis (CGN) groups. Plasma PMPs,
P-selectin, EMPs and vWF in stage I-II CKD patients were
significantly higher than those of stage III-V CKD patients,
no significant difference was found within stage I-II CKD
patients or stage III-V CKD patients. PMPs and EMPs
were positively correlated with blood pressure and 24-hour
urinary protein, but no significant correlation was found
with the creatinine clearance rate, hemoglobin or cholesterol. P-selectin and vWF were positively correlated with
PMPs and EMPs respectively. Conclusion: CKD patients
have significant platelet activation and endothelial dysfunction, which was involved in CKD’s occurrence and development; high blood pressure and protein urea are important
causes for platelet activation and endothelial dysfunction in
patients with CKD; PMPs and EMPs can be used as new
markers for dysfunctional platelet activation and
endothelium.
Po38. A potential role of mir-210 in the
regulation of angiogenesis after stroke
Abstracts
Po39. Personalized evaluation of
chemotherapeutic sensitivity and survival
in platinum- based chemotherapy
Yimin Zhu
Deptment Epidemiology and Biostatistics, School of Public Health,
Platinum-based chemotherapy regimens are the mainstay
treatment for the cancers such as lung, colorectal, ovary
cancers. The five-year survival rate remains at low level,
for example, only 15% for lung cancer. This clinical strategy is limited by the development of chemoresistance and
toxicity. However, at present, only clinical variables such
as stage, location, and performance scores are used to
guide treatment decisions for the cancer patients. There are
great differences in sensitivity and toxicity in the patients
even with same-stage disease. Therefore, an effective
evaluation to the response to the clinical treatment is
important and very useful in clinical decision-making. The
annotation of human genome provides an opportunity to
explore the impact of genetic variation in determining the
risk and prognosis differences in complex diseases. The
genome, through the information imprinted in its DNA
sequence, is an important determinant of biologic phenotypes of host in chemical absorption, transportation,
metabolism, DNA repair, cell cycle, and apoptosis, etc.
The variations of functions in the key genes in the
platinum- based pathway might collectively make an
important contribution to the different response to chemotherapeutic sensitivity and survival. Single nucleotide
polymorphisms (SNPs), copy number variations (CNVs),
DNA repair capacity (DRC), which can be detected with
non-invasive methods, are useful potential biomarkers in
the personalized evaluation in platinum- based
chemotherapy.
Po40. Atrogin-1 suppressed autophagy
induced by LPS in cardiomyocytes
Junjie Li and Huihua Li *
Department of Pathology and National Laboratory of Medical
Molecular Biology, Institute of Basic Medical Sciences, Chinese
Academy of Medical Sciences and Peking Union Medical College,
Beijing, China.
Autophagy is a lysosomal degradation pathway and an
important player in many critical biological processes such
as cellular response to starvation, infections, cell survival
and death, muscle atrophy, cancer, and host defense.
Atrogin-1/MAFbx is a muscle-specific ubiquitin E3-ligase
required for muscle atrophy. It is unclear whether
Atrogin-1/MAFbx could independently regulate autophagy
induced by LPS in cardiomyocytes. Our aim is to study
the effect of atrogin-1/MAFbx in regulation autophagy
induced by LPS in cardiomyocytes for later research of
the mechanism of atrogin-1controls autophagy and its
possible roles in the causation and prevention of heart diseases. Cardiomyocytes were isolated and cultured by enzymatic disassociation of 1- to 2-day-old Sprague-Dawley
rats, Cardiomyocytes were infected with Ad-GFP or
Ad-atrogin-1-GFP for 24 hours and were stimulated with
LPS (100 ng/ml), fixed, immunolabeled with LC3 antibody followed by Alexa Fluor 568-conjugated goat antirabbit IgG (red,), and stained with DAPI to visualize the
nuclei (blue), Autophagosomes were quantified by counting LC3-positive dots and normalizing for cross-sectional
area, quantitation of the percentage of cells with autophagosomes with image-pro plus 6.0 software; total RNA was
prepared, Quantitative realtime RT-PCR analysis was performed in triplicate using specific primers of rat LC3,
Atg12, Atg4b, Bnip1, Bnip3 and vp34. Immunoblotting
for LC3 was performed with 5 mg of cell extracts. LPS
induced a significant increase in the percentage of cells
expressing autophagosomes. Autophagosome formation
determined by real time PCR. EphrinA3 mRNA and
protein expression in ischemic cortex of different groups
were detected by RT-PCR and immunofluorescence
respectively. At last, microvessels in endothelial cells were
demonstrated by immunofluorescent staining for factor
VIII and the microvessel density (MVD) in different
groups was counted. Real-time PCR results showed that
mir-210 expression was significantly up-regulated after
brain ischemia, as 7.2 + 0.56 (1 d), 20.1 + 0.87 (3 d) and
20.3 + 0.76 (7 d) folds changes compared with sham
group. While RT-PCR showed a gradually increased
mRNA expression of ephrinA3 in ischemic cortex, a
higher percentage of ephrinA3-positive cells of sham group
(66% + 0.55%) was detected by immunofluorescence,
compared to 1 d (45% + 0.38%), 3 d (36% + 0.65%) and
7 d (27% + 0.28%) post-ischemia groups, indicating a
mir-210-mediated post-transcriptional regulation of
ephrinA3 protein expression. Immunofluorescence of VIII
factor showed a gradually increase of MVD after stroke, as
2.85 + 0.51 (1 d), 4.47 + 0.44 (3 d), 5.63 + 0.69 (7 d)
compared with 2.79 + 0.21 (sham group). These results
suggest that mir-210 may play a critical role in angiogenesis after stroke.
Abstracts
induced by LPS is suppressed by the overexpression of
atrogin-1. Overexpressed atrogin-1 prevented the increase
in transcript levels of LC3, Atg12, Atg4b, Bnip1, and
vp34 induced by LPS. Overexpression of atrogin-1
decreased the amount of LC3-I and blocked the conversion of LC3-I to LC3-II after LPS stimulation. Here we
find that LPS leads to the activation of autophagic pathways in cardiomyocytes and the induced- autophagy is
suppressed by the overexpression of Atrogin-1.
Po41. Association between
polymorphisms of estrogen motablic
enzymes and susceptibility to colorectal
cancer
Department of Hygienic Toxicology, College of Preventive Medicine;
Key Lab of Medical Protection for Electromagnetic Radiation,
Ministry of Education of China, Third Military Medical University,
Chongqing, China
Fax: þ86-23-68752589; E-mail: [email protected]
Both experimental and epidemiologic studies have
clarified that estrogen and its metabolic enzymes play
important roles in carcinogenesis of colorectal cancer,
suggesting that some genotypes of estrogen metabolic
enzyemes could be candidate cancer susceptibility genes.
To validate the hypotheses, we investigated the association of the polymorphisms in estrogen-metabolizing
enzymes genes with susceptibility to CRC in this casecontrol association study.
The case-control study included 433 patients with CRC
and 790 control subjects. All CRC patients were unrelated
ethnic adult Chinese and residents in Chongqing municipality and its surrounding regions, excluding FAP and
HNPCC patients, treated at 3 affiliated hospitals of the
Third Military Medical University. All control subjects
were patients which were enrolled due to trauma with no
signs of malignancies. At recruitment, informed consent
was obtained from each subject, and personal information
on demographic factors, medical history, tobacco and
alcohol use, and family history of CRC were collected
with structured questionnaire.
Genotyping was performed by SNPlex assay for
rs2486758 in CYP17, rs10046 in CYP19, rs676387 in
HSD17B1, rs4680 in COMT. The data were collected
with Data Collection and analyzed with Genemapper.
Yanhong Zhou, Huan Yang, Ziyuan Zhou, An Hui,
Zhihong Cui, and Jia Cao *
The Hardy-Weinberg equilibrium at each SNP site in
control groups was analyzed with fit x2 test.
Unconditioned- Logistic regression analysis was used for
the calculation of Odds ratio and 95% confidence interval, adjusted with sex, ages, and smoking and alcohol
status. SAS astatistical package 9.0 and UNPHASE 3.07
were used to conduct the analyses. Analyes included evaluating the distribution of the alleles, genotypes, haplotypes and diplotypes in the study population, the
independent associations of genetic polymorphisms with
colorectal cancer risk, and the joint effect of genotypes on
colorectal cancer risk.
We carried out multiple hypotheses testing using the
Benjamini-Hochberg method to control the false discovery rate (FDR) in the unconditional logistic regression
analysis.
Haplotypes
were
estimated
using
Haploview4.1.MDR and conditioned-Logestic regression
analysis were used for gene-gene interaction analysis.
Conditioned-logistic regression analysis was used for
genetic-environmental interaction. Two-side test was used
for all the analysis.
rs676287 in HSD17B1 and rs10046 in CYP19 were
found to be associated to CRC risk after FDR analysis.
For rs676387 in HSD17B1: GT vs GG: OR ¼ 3.280,
95%CI ¼ 2.326 4.625; TT vs GG: OR ¼ 3.018,
95%CI ¼ 2.109 4.318, TT þ GT vs GG: OR ¼ 2.002,
95%CI ¼ 1.330 3.013. There were gene-environment
interaction between rs676387 and age, gender, smoking
and drinking.
For rs10046 in CYP19: CT vs CC: OR ¼ 0.594,
95%CI ¼ 0.442 0.797; TT þ CT vs CC: OR ¼ 0.638,
95%CI ¼ 0.480 0.849. There were gene-environment
interaction between rs10046 and age, sex, smoking and
drinking.
There was no association between CRC risk and polymorphisms at rs2486758 in CYP17 or rs4680 in COMT .
Gene-gene interactions exist between the following pairs
of SNPs: (1) CYP17 rs2486758 and CYP19 rs10046; (2)
HSD17B1 rs676387 and CYP19 rs10046; (3) ERa
rs2071454 and CYP19 rs10046; (4) ERa rs2071454 and
HSD1B1 rs676387; (5) ERa rs2077647 and HSD1B1
rs676387; (6) ERa rs2077647 and CYP19 rs10046.
Two SNPs, rs676287 and rs10046 in HSD17B1 and
CYP19, respectively, have correlation with CRC susceptibility.
Gene-gene interactions lie in rs2486758 of CYP17,
rs10046 of CYP19 and rs676387 of HSD17B1. [This work
was supported by General Program (No. 30700676,
No.30800933) grants from the National Natural Science
Foundation of China (NSFC)]
Abstracts
Po42. Stability analysis on liver cancer
related miRNA in serum
Yan Li 1, Zhenggang Jiang 2, Lijian Xu 1, Hu Yao 1,
Jiangfeng Guo 1 *, and Xianfeng Ding 1 *
1
College of Life Science, Zhejiang Sci-Tech University, Hangzhou,
China
2
The Center for Disease Control and Prevention of Zhejiang Province,
Hangzhou, China
Fax: þ86-5718684-3303; E-mail: [email protected] (X. D.);
Tel: þ86-571-86843302; Fax: þ86-571-8684-3303; E-mail: jfguo@
zstu.edu.cn (J. G.)
Po43. Erythropoietin attenuates acute
exhaustive exercise
Xixiu Lin
Hunan Normal University, Changsha, China
Erythropoietin (Epo) was recently defined as an
endogenous agent with more than hematopoietic functions,
and has been shown to have a protective effect against the
tissue. The study was to investigate this effect on renal
injury of the rat induced by acute exhaustive exercise.
Twenty-four adult healthy male SD rats were randomly
divided into normal control group (C), exhaustive exercise
test group (ET) and ET plus EPO pre-treated group (ET þ
EPO). Serum urea nitrogen and creatinine; urine protein;
nitric oxide (NO), and nitricoxide synthase (NOS) activities
were
measured.
Kidney
was
evaluated
by
Electronmicrograph. Cell apoptosis was identified by
Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick
End Labeling (TUNEL) assay. Compared with controls,
the animals in the ET group revealed a significant increase
in serum urea nitrogen, serum creatinine, urine protein; and
a significant decrease in renal tissue NO and NOS activity.
Electronmicrograph showed a clear damage in the filtration
membrane of glomerular with cell apoptosis and necrosis
in the rats of ET group. TUNEL assay revealed a remarkably higher apoptotic index in ET group, compared
with the controls (P , 0.01). Compared with ET group,
MicroRNAs (miRNA) are non-coding, single-stranded
RNAs of 22 nucleotides and constitute a novel class of
gene regulators that are found in both plants and animals.
Recent evidence has shown that miRNA mutations or aberrantly expression correlate with various human cancers and
indicated that miRNAs can function as tumour suppressors
and oncogenes. While many studies have focused on
miRNA expression in physiological and pathological processes, variables related to miRNA for new serum biomarkers have simultaneously emerged. Now miRNA has
been applied to early detection of cancer and monitoring of
cancer recovery by using detection of peripheral blood.
Up to now, there are no reports regarding liver cancer
specific miRNA biomarkers in serum for the great threat of
liver cancer to human life. Therefore a systemic research
on the characteristic of miRNA is quite necessary.
Liver cancer Huh-7 cell-line, liver tumor tissues and
clinical serum samples were performed in the role of experiment material. A systemic treatment, such as different
temperature (2808C, 2208C, 48C, room temperature and
378C) treated for 3h, in room temperature treated for 0, 1,
3, 6, 12, 24 h, RNase A treated for 0, 3, 6, 12 h incubation
in 378C, DNase I treated for 0, 3, 6, 12 h incubation in
378C, different free-thaw cycles (0, 2, 5, 7, 10 cycles)
treated, different pH value (control, pH ¼ 1, 6, 9, 13) of
solution treated for 3h incubation in 378C were performed
before quantitative reverse transcription-polymerase chain
reaction (qRT-PCR) analysis with 40 cycles. Furthermore,
liver cancer related miRNAs were detected in each reaction.
We used 18S rRNA as control gene. All the results indicated that 18S rRNA was fragile for decreased relative
expression sharply. MiRNAs could be resistant to harsh
conditions simultaneously. The P-value indicated the
repeatability of the data by using the Student’s t-test. P ,
0.05 was considered to be significant.
Meanwhile, we evaluated the Pearson’s correlation coefficient of liver cancer-related miRNAs expression of 22
healthy human subjects by qRT-PCR. Expression levels of
serum miRNAs were reproducible and consistent among
22 healthy human subjects because the R value was access
to 1, and P , 0.05. The result was considered to be significant. Pearson correlation scatter plot of the relative serum
miRNAs expression between male and female of R2 was
0.0953. Results suggested that miRNA expression is not
correlated between genders.
Taken together, these results implied that liver cancer
related miRNA (miRNA-21, -25, -29c, -93, -198, -221,
-222) expression levels in serum were quite stable, also
present and detectable, reproducibly consistent among individuals of the same species in serum. They will be potential for serum liver cancer biomarkers in future.
On the other side, it is difficult to obtain abounding and
high-quality of RNA in serum. Our tests has shown that
pre-heating procedure is a robust serum RNA extraction
method and efficient for RNA isolation. This method is
also essential for further serum source microRNA study. In
fact, after qRT-PCR, the CT (threshold cycle) value
decreased at least 5. In conclusion, based on our study, preheating provided an optimized protocol for serum RNA
isolation.
Abstracts
ET þ EPO group had a significant lower level of urine
protein and apoptotic index and significant higher level of
renal tissue NO and NOS activity. The pathological
changes were much less in the kidney of ET þ EPO group.
Moreover, EPO decreased the exercise-elevated apoptotic
index (P , 0.05). EPO intervention prevented the renal cell
apoptosis and counteracted the exhaust induced low NO,
NOS contents, thus protected against the acute renal injury.
EPO would be a potential drug in preventing the acute
renal injury after exhaustive exercise.
Po44. Analysis of Fas and Fas ligand
expression and function in lung cancer
cell lines
Chunhua Ling 1,
Shiying Zheng 2*,
Dong Jiang 2,
and
1
Department of Medicine, the First Affiliated Hospital of Suzhou
2
Department of Thoracic Surgery, the First Affiliated Linical Hospital
of China Medcial University, Shenyang, China
Fas ligand (FasL) and its receptor (Fas, CD95) are a
set of regulatory components in immune system.
Fas-FasL ligation results in the apoptosis of cells, which
was involved in the destruction of activated T lymphocytes and play a critical role in the maintenance of
immunological homeostasis and peripheral tolerance. It
has also been reported that chemotherapy-induced apoptosis is not dependent on Fas-FasL interactions. We therefore investigated the expression of Fas and Fas ligand
(FasL) lung cancer cell lines and evaluated the significance of these molecules in chemotherapy treatment.
Human lung cancer cell lines, NCI-H157, NCI-H322,
NCI-H460, NCI-H1299, NCI-N417, EBC1, PC9, A549
and LK2 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/
streptomycin in a humidiffied incubator with 5% CO2 at
378C. Immunoblotting, RT- PCR and flow cytometric
analyses were carried out to measure the expression of
Fas and FasL, in order to examine their interactions and
effects on cell growth and apoptosis.
Fas and FasL were co-expressed in most of the cell
lines but to varying degrees. There was no correlation
between Fas and FasL expression. Fas-ligation using
agonistic anti-Fas antibody induced apoptosis in cells.
This process was significantly correlated with Fas
expression (P ¼ 0.0075). Blockade of the Fas-FasL
system had no effect on the proliferation of cancer cells.
Po45. MicroRNAs associated with renal
angiogenesis after renal ischemia/
reperfusion injury in mouse
Fen Liu, Jue Wu, Yuanlei Lou, Qiongfang Ruan, An Xie,
Yang Yang, Suping Cui, and Yang Wang *
MicroRNAs (miRNAs) are a class of endogenous, conserved, small non-coding RNAs which play important
regulatory roles in many physiological processes. Here we
sought to investigate potential involvement of miRNAs in
renal ischemia/reperfusion (I/R) injury and renal angiogenesis in mouse.Male Balb/c mice were subjected to a standard renal I/R to induce acute kidney injury (AKI) after
45 min of bilateral renal artery clamping. Following 24 h
of I/R or sham operation, kidey tissues were collected and
subjected to detect the changes of microRNAs expression
through Agilent microRNA microarrays.
Seventy-six miRNAs exhibited more than two-fold
changes in their expression level after kidney I/R injury.
Among them, 40 miRNAs decreased and 36 miRNAs
increased. The results were validated by quantitative realtime RT-PCR. Renal ischemic injury significantly
increased miRNA-210, miRNA-126 and miRNA-92a
expression, with prominent changes at 4 h and 24 h after
recovery.(n ¼ 3; P , 0.05).Computer prediction analysis
indicated that some of the target genes might be directly
associated with signal pathways relevant to angiogenesis.
This is the first report about miRNAs changes in response
to kidney I/R in mouse. Our results imply that miRNAs
may be involved in the regulation of angiogenesis
processes after renal I/R injury. Further studies are
required to understand the mechanisms of miRNA-based
angiogenesis.
Jinfeng Ge 2
Cisplatin induced apoptosis in cancer cells and upregulated Fas (7 of 11, 64%) and FasL (9 of 11 82%)
expression. However, cisplatin-induced apoptosis was not
suppressed by the antagonistic anti-FasL antibody.
Our data indicated that Fas and FasL were
co-expressed in lung cancer cell lines. Fas ligation is
functional in the induction of apoptosis, which was
dependent on the levels of Fas expression. In contrast,
Fas-FasL interactions appeared to be non-functional in
cisplatin-induced apoptosis of cancer cells. It suggested
that besides Fas and FasL, there exist unknown intracellular factors may regulate apoptosis in cancer cell lines and
play major roles in chemotherapy.
Abstracts
Po46. MiR-210 up-regulates Notch
signaling for capillary formation in
hMECs
Yuanlei Lou 1, Zhifeng Deng 2, Faliang Gao 2, An Xie 1,
Fei Guo 1, Qiangfang Ruan 1, Yang Yang, and
Yang Wang 1 *
1
2
Po47. Identification of microRNAs
responded to adenovirus type 3 infection
in human laryngeal epithelial cells by
SOLid system
Yuhua Qi §, Xiling Guo §, Lunbiao Cui, Zhiyang Shi *,
Tao Wu, Yunfeng Shan, Yiyue Ge, Jun Shan, and
Hua Wang
Key Lab of Enteric Pathogenic Microbiology, Ministry of Health,
Institute of Pathogenic Microbiology, Jiangsu Center for Disease
Prevention and Control (CDC), Nanjing, China
§
Contributed equally to this work.
Previous work had shown that adenovirus infection can
cause various illness depending on the infecting serotype,
such as gastroenteritis, conjunctivitis, cystitis, and rash
illness, but the molecular mechanisms of the host response
to adenovirus infection are still not completely understood.
MicroRNAs (miRNAs) have been reported to play essential
roles in cell proliferation, cell differentiation, and pathogenesis of human diseases including viral infections. MiRNAs
have also been reported as a tool for gene specific therapeutics against viral infections. To determine which
miRNAs play roles in the host response to adenovirus infection, we analyzed the miRNA expression profiles from adenovirus type 3 (AD3) infected Human laryngeal epithelial
(Hep2) cells using a SOLiD deep sequencing.
Hep2 cells were cultivated in complete RPMI medium
1640 and infected with AD3 under 50% tissue culture
Angiogenesis in the adult organism is a complex multistep process involving degradation of extracellular matrix,
migration and proliferation of endothelial cells, initiation of
sprouting, lumen formation and establishment of blood flow.
Many genes expressed in endothelial cell are involved in
these events by affecting different intracellular signal pathways during angiogenesis. Notch pathway is known to play
key roles in embryonic vascular development and postnatal
angiogenesis. Notch signaling is an extremely evolutionary
conserved cell signaling system which controls cell fate
decisions in metazoans through local cell-cell interactions.
Mammals possess four different Notch receptors (Notch1 to
Notch4) and five ligands (Dll 1, Dll 3, Dll 4, Jagged-1and
Jagged-2). Interaction of Notch receptors with their ligands
leads to cleavage of the Notch intracellular domain (NICD)
which migrates into the nucleus and activates transcription
of primary target genes of Notch signaling. It is reported
that Delta-like ligand 4 (Dll4)-Notch signaling induced by
VEGF determines the initiation and stabilization of angiogenic sprouting.
Recently, a few specific microRNAs (miRNAs) that regulate endothelial cell functions have been described. MiR-210
is amicroRNA thatcan be induced by hypoxia in all tested cell
types. Recent research indicates that miR-210 plays a crucial
role in regulating biological processes of blood vessel endothelial cell in response to hypoxia. MiR-210 affects cell survival, migration and differentiation by down-regulation of
mRNA enphrinA3 expression. Up-regulation of miR-210 in
endothelial cells in the situations of hypoxia stimulates the formation of capillary-like structures on matrigel. In our previous
study, we have constructed a recombinant lentiviral expression
vector of miR-210 which can be effectively transfected into
human microvascular endothelial cells (hMECs). In vitro
capillary-like formation assay has shown that hMECs plated
on Matrigel expressing lentiviral recombination pre-miR210
significantly increased tubulogenesis. The result suggested
that hypoxia-trigged miR-210 expression affects various biological processes inblood vessel endothelial cells. However,
the role of miR-210 in Notch pathway remains unclear.
The present study is to investigate whether miR-210 is
involved in the regulation of notch signaling during
neovascularization. hMECs were transfected with lentiviral recombination pre-miR210. Quantitative real time
RT-PCR was performed to evaluate miR-210 expression
in hMECs. The enphrinA3 protein expression was
measured by immunofluorescence. Notch1 gene and
NICD protein expression was detected by RT-PCR and
western blot respectively. The results of real-time quantitative RT-PCR showed that the miR-210 level incells
transfected with lentiviral recombination pre-miR210 was
increased significantly compared with the control cells
(P , 0.05). Immunofluorescence analysis showed that
enphrinA3 labeled with FITC was located in the cytoplasm, and was decreased obviously in lentiviral recombination pre-miR210 -transduced cells. The results of
RT-PCR and western blot showed that the expressions of
Notch1 mRNA and NICD protein in cells transfected
with lentiviral recombination pre-miR210 were significantly increased compared with that in control cells (P ,
0.05). Our data indicates that overexpression of miR-210
can up-regulate Notch signaling in hMECs, which could
contribute to hypoxia-induced angiogenesis
Abstracts
Po48. Silica nanoparticles as carriers in
the controlled release of florfenicol
Meirong Song 1*, Aimin Ning 1, Yanyan Li 2,
Shaoming Fang 2, and Baoan Cui 1*
1
Henan Agricultural University, Zhengzhou, China
Department of Education, Henan Institute of Science and
Technology, Xinxiang, China
3
Key laboratory of surf ace and interface science, Zhengzhou
University of Light Industry, Zhengzhou, China
Fax: þ86-371-63558130; E-mail: [email protected] (M.S.);
E-mail: [email protected] (B. C.)
2
The aim of this study was to use silica nanoparticles as
the carrier to load florfenicol in aqueous solution.
Florfenicol, which was selected as a model molecule since
it is a broad-spectrum antibiotic drug with poor solubility
in water, was absorbed on silica nanoparticles in aqueous
solution through a natural cooling process from 908C to
room temperature. Tween-80 was added as a stabilizer to
limit the growth of drug loaded particles. The florfenicol
-loaded silica particles was characterized by transmission
electron microscopy, zetasizer laser particle size analyser,
Fourier transform infrared spectrum, thermal gravimetric
analysis and ultraviolet–visible light spectroscopy. The
results show that florfenicol was adsorbed by silica nanoparticles without degradation at a loading of 28.92 wt%; in
contrast to the rapid release from pure florfenicol, the
drug-loaded silica particles showed a slower release over a
longer time.
Po49. Effects of Niacin on C57BL6J mice’
adipose tissue, serum lipid and
atherosclerosis
Zhongqun Wang and Naifeng Liu
Department of Cardiovascular Medicine, Zhongda Hospital, Southeast
University, Nanjing, China
Our aims are to investigate the effects of niacin on
C57BL/6J mice adipose tissue, serum lipid and atherosclerosis, and to explore the potential relationships among them.
Total 28 male C57BL/6J mice were randomly divided into 3
groups–control group (n ¼ 8), model group (n ¼ 10), niacin
treatment group (n ¼ 10), respectively fed with normal diet,
high cholesterol diet, high cholesterol diet þ 1% (w/w)
niacin. After 14 weeks, serum lipid level (total triglyeride,
total cholesterol, HDL cholesterol, LDL cholesterol and
apoA-I) was measured by enzymatic method and by immunoturbidimetery. Lesions of aortic arteries were stained with
hematoxylin eosin. The content of cholesterol in arterial wall
and subcutaneous adipose tissue in groin was quantitated by
high performance liquid chromatography. The expression of
LXRa, ABCA1 and ABCG1 mRNA in subcutaneous
adipose tissue was determined by RT-PCR. The content of
cholesterol in subcutaneous adipose tissue in control group,
model group, niacin treatment group was 3.13 + 0.19,
20.81 + 1.97 and 4.00 + 0.81 mg/g, respectively. Compared
with model group, niacin treatment could increase the
expression of LXRa, ABCA1 and ABCG1 mRNA 144%,
47.3% and 73.8% respectively. And furthermore, it could
also downregulate the level of serum total triglyeride, total
cholesterol and LDL cholesterol, upregulate the level of
serum HDL cholesterol, apoA-I. Pearson correlation analysis
showed that there was a positive relation between the content
of cholesterol in arterial wall and the ratio of intima/media
infectious doses. At 6 h, 24 h, 48 h, 72 h post infection
( p.i.), RNA samples were prepared from AD3 infected and
controlled Hep2 cells using a mirVana miRNA Isolation Kit
(Ambion). RNA samples isolated from cells after 72 h p.i.
were processed into SOLiD deep sequencing and the results
were confirmed by Q-PCR. Furthermore, the expression
levels of candidate miRNAs at different time points after
AD3 infection were measured by Q-PCR.
SOLiD results showed that 492 precursor miRNAs were
identified in the AD3 infected Hep2 cells, and 540 precursor
miRNAs were identified in the controls. A total of 44
miRNAs demonstrated high expression and 36 miRNAs
showed lower expression in the AD3 infected cells than controls. Some candidate miRNAs expression levels were confirmed by Q-PCR analysis. To further understand the
miRNAs expression in AD3 infected Hep2 cells at 6 h,
24 h, 48 h, and 72 h p.i., we performed Q-PCR experiments
on candidated miRNAs. The ratio of miRNAs in AD3
infected cells versus controls was calculated by using the
equation 22DDCT. In fact, our results indicated that the
miRNAs expression shows complicated variation at these
time points after infection with AD3. For example,
miR-27b, miR-125b, and miR-101 showed up-regulated
expression in AD3 infected cells comparing control cells
at 6 h post infection. However they showed down-regulated
expression in AD3 infected cells at 72 h p.i.. MiR-17
expression was up-regulated at 6, 24, and 72 h p.i. in AD3
infected cells. But at 48 h p.i., miR-17 showed downregulated expression in AD3 infected cells.
SOLiD sequencing provides a useful method for identification of the miRNAs profiles in AD3 infected Hep2 cells.
Our findings illustrate the overall host response to AD3 infection and will aid in understanding the host response to this
virus. Further studies are required to identify the miRNA
target genes and the functions of the miRNAs in the complex
molecular network regulation during the virus infection host
cells using bioinformatics tools.
Abstracts
thickness (r ¼ 0.58, P,0.05). In addition to those above,
niacin treatment could thin the intima thickness, decrease the
ratio of intima/media thickness and downregulate the subendothelium lipid-accumulation, especially cholesterolaccumulation. Niacin treatment may promote the reverse
cholesterol transport of peri-adipose tissue, bring the
changes of serum lipid profile, and furthermore influence the
subendothelium lipid-accumulation, especially cholesterolaccumulation, and subsequently reverse the aortic
atherosclerosis.
Po50. Mechanisms of IFN-g affecting
lipid accumulation in THP-1
macrophages
Zhongqun Wang and Naifeng Liu
Our aim is to investigate the mechanisms of IFN-g affecting lipid accumulation in THP-1 macrophages. THP-1
macrophages were divided into 4 groups, A (control group):
serum-free medium with 2g/L BSA; B: serum-free medium
with 2 g/L BSA þ 50 mg/L IFN-g; C: serum-free medium
with 2 g/L BSA þ 50 mg/L IFN-g þ 25 mg/L oxLDL; D:
serum-free medium with 2 g/L BSA þ 100 mg/L IFN-g þ
25 mg/L oxLDL. After 24 h, intracellular lipid accumulation
was measured by oil-red O staining and high performance
liquid chromatography; subcellular localization and cytomembrane activity of PKC were observed by FITC immunofluorescent
staining
and
PepTagwAssay
for
Non-Radioactive Detection, respectively; the mRNA level
of CD36 and ACAT-1 were determined by reverse
transcription-polymerase chain reaction (RT-PCR). IFN-g
could induce significantly the accumulation and fusion of
lipid droplet. The number of oil-red O-positive cells in 100
macrophages in group A, B, C and C was 8 + 1, 21 + 3,
35 + 2, 69 + 6, respectively; the ratio of CE/TC in four
groups was 22.5%, 40.4%, 49.6%, 71.2% (higher than 50%,
having been foam cells), respectively; there was significantly difference among them. Along with the increasing of
IFN-g concentration, FITC-labeled fluorescence intensity
became stronger significance; furthermore, stronger fluorescence obviously moved from cytoplasm to cytomembrane, that is to say, the activity of PKC in macrophages
became significantly strong. RT-PCR experiment indicated
that IFN-g could downregulate the expression of CD36, but
upregulate ACAT-1. IFN-g may induce PKC pathway and
promote the expression of ACAT-1, and subsequently
increase the cholesterol-uptake, -synthesis and -esterification
of macrophages loaded with or without oxLDL.
Jue Wu, Fen Liu, Qiongfang Ruan, An Xie, Yuanlei Lou,
Suping Cui, Jun Deng, Yang Yang, and Yang Wang *
Institute of Urology, the First Affiliated Hospital of Nanchang
University, Nanchang, China
Renal ischemia/reperfusion (I/R) injury is in relation
with renal transplantation and renal operation. As an important pathological process, the timely reconstruction of renal
blood flow in ischemic region is a key treatment for renal
ischemic injury. Animal experiments and clinical studies
showed that compensatory angiogenesis was increased after
issues ischemia. Most studies have confirmed that the
VEGF pathway signalling has a critical regulatory role in
endothelial cell proliferation, differentiation, migration and
angiogenesis and is involved in the regulation of
ischemia-induced angiogenesis in tissue ischemia, which
are perhaps the most important mechanism in regulation of
angiogenesis in renal ischemia. Recently, studies have
showed that microRNAs (miRNAs) are small noncoding
RNAs and regulate gene expression at the posttranscriptional level by either degradation or translational
repression of a target mRNA that regulate physiological
and pathological processes. And a few specific miRNAs
targeting endothelial cell function and angiogenesis in
tissue ischemia-induced have been identified. Our results
showed that some of miRNAs expression changes after
renal ischemia may be involved in targeting VEGF
pathway signaling to regulate angiogenesis.
We investigated the expression changes of miRNAs in
mouse ischemic renal. These mice were divided into two
groups, which are post-ischemia group and sham group.
Mice were subjected to a standard renal I/R to induce acute
kidney injury (AKI) after 45 min of bilateral renal artery
clamping. Mice were killed at 4, 24, 48, 72 h after ischemia, and the impaired tissues were extracted for examine.
The sham group was not operated as controls. The
expression of CD31 was examined in frozen tissue sections
by immunofluorescence staining, and the microvessels in
ischemic tissues of each group were counted. MiRNAs
expression changes were determined by quantitative realtime RT-PCR analysis at 4 and 24 h following I/R. VEGF
and Flk-1 mRNA expression changes were examed by
using quantitative real-time RT-PCR analysis at 4 and 24 h
following I/R. Flk-1 protein expression changes were
detected by western blot analysis at 24 and 72 h following
I/R.
The immunofluorescent staining results of CD31 showed
a significant increase of microvessels in ischemic region in
Department of Cardiovascular Medicine, Zhongda Hospital, Southeast
University, Nanjing, China
Po51. MicroRNAs targeting VEGF
pathway induced angiogenesis after renal
ischemia reperfusion
Abstracts
Po52. CpG island promoter
hypermethylation of the pro-apoptotic
gene caspase-8 is a common hallmark of
relapsed glioblastoma multiforme
Feng Xu, Jiangang Liu, Shiming Zhang, and
Shiying Zheng *
The First Affiliated Hospital of Suzhou University, Suzhou, China
*Correspondence address. þ86-512-65263570; E-mail: Syzheng88@
sina.com
Glioblastoma multiforme (GBM) is an incurable malignancy with inherent tendency to recur. In this study we
have comparatively analyzed the epigenetic profile of
thirty-two paired tumor samples of relapsed GBM and
their corresponding primary neoplasms with special attention to genes involved in the mitochondria-independent
apoptotic pathway.
The CpG island promoter hypermethylation status was
assessed by methylation-specific PCR and selected samples
were double-checked by bisulfite genomic sequencing.
Thirteen genes were analyzed for DNA methylation: the
pro-apoptotic genes CASP8, CASP3, CASP9, DcR 1, DR4,
DR5 and TMS1; the cell adherence genes CDH1 and
CDH13; the candidate tumor suppressor genes RASSF1A
and BLU; the cell-cycle regulator gene CHFR; and the
DNA repair MGMT gene.
The CpG island promoter hypermethylation profile of
relapsed GBM in comparison with their corresponding
primary tumors was identical in 37.5% of cases, while in
62.5% of patient differences in the DNA methylation patterns of the thirteen genes observed. The most prominent
distinction was the presence of previously undetected
CASP8 hypermethylation in the GBM relapses (P ¼
0.031). This finding was also linked to the observation that
an unmethylated CASP8 CpG island together with methylated BLU promoter in the primary GBM was associated
with prolonged time to tumor progression (P ¼ 0.0035).
Our data strongly suggest that hypermethylation of
pro-apoptotic CASP8 is a differential feature of GBM
relapses.
These findings may foster the development of therapeutic approaches using DNA demethylating drugs and
activators of the extrinsic apoptotic pathway to improve the
dismal prognosis of GBM
Po53. Genetic polymorphisms in
progesterone receptor gene and
susceptibility to hepatocellular carcinoma
Xiaoyan Yuan 1, Yun Zhai 2, Jia Cao 1, Xiumei Zhang 2,
Ling Yu 2, Gangqiao Zhou 2 *, and Fuchu He 2 *
1
Department of Hygienic Toxicology, Preventive Medical College,
Third Military Medical University, Chongqing, China
2
The State Key Laboratory of Proteomics, Institute of Radiation
Medicine, Beijing Academy of Military Medical Scienees, Beijing,
China
*Correspondence address. Tel: þ86-10-68177417; E-mail: hefc@nic.
bmi.ac.cn (F. H.); Tel: þ86-10-68177417;
E-mail: [email protected] (G. Z.)
Hepatocellular carcinoma (HCC) is one of the most
common malignancies in the world. Human epidemiologic
studies have documented that oral contraceptives (OCs) can
increase the susceptibility to HCC. Most OCs contained a
progestin in combination with an estrogen. As we known,
both estrogen and progestin exert their effects by binding
to their receptors. So polymorphisms of estrogen receptor
(ERs) and progesterone receptor (PR) genes may have
bearing on the risk of HCC. Recently, we observed a significant association between the risk of HCC and the polymorphisms of the ERa (ER1) gene. In this study, we
investigated the association between the polymorphisms in
PR gene and the risk of HCC in a Southern Chinese population. This case-control study consists of 434 incident
patients with HCC and 480 control subjects. Two polymorphisms in PR gene were genotyped by polymerase
renal ischemia compared with control group. Real-time
RT-PCR analysis showed that ischemic kidney injury significantly increased miRNA-210, miRNA-320, miRNA-126
and miRNA-92a expression compared with sham controls,
with prominent changes at 4 and 24 h after recovery. mRNA
and protein expression changes of VEGF signaling moleculars were detected by real time RT-PCR and western blot
analysis respectively. The expression of VEGF and Flk-1
mRNA in I/R injury was gradually upregulated after ischemia. Western blot analysis showed that Flk-1 protein
expression was increased in I/R injury at 24 and 72 h compared with the sham controls.
We concluded that renal I/R to induce acute kidney injury
(AKI) significantly increased miRNA-210, miRNA-320,
miRNA-126 and miRNA-92a expression with prominent
changes at 4 and 24 h after recovery. And compensatory
angiogenesis was increased significantly after renal ischemia. VEGF and Flk-1 mRNA expression were increased in
I/R injury compared with the sham controls and Flk-1
protein expression were also increased in renal I/R injury
compared with the sham controls. These results provided
evidence that miR-210, miRNA-320, miRNA-126 and
miRNA-92a may be involved in the mechanisms of renal
ischemic injury disease through targeting VEGF pathway to
regulate angiogenesis.
Abstracts
Po54. Expression of elastin, LOX, or
elafin in the cardinal ligament of pelvic
organ prolapse
Shiqian Zhang 1 *, Linlin Zhang 2, Hao Yu 3, Yanlei Dong 1,
and Haiqing Lai 1
1
Department of Obstetrics and Gynecology, Qilu Hospital of
Shandong University, Ji’nan, China
2
Department of Obstetrics and Gynecology, Fourth People’s Hospital
of Ji’nan, Ji’nan, China
3
Department of Gynecologic Oncology, Shandong Cancer Hospital
and Institute, Ji’nan, China
*Correspondence address. Tel: þ86-531-82169577; E-mail:
[email protected]
The precise mechanism of pelvic organ prolapse (POP)
is not well known. We investigated the expression of
elastin, lysyl oxidase (LOX), and elafin in cardinal ligament in the pathogenesis of pelvic organ prolapse (POP).
The study included 60 subjects with POP and 60 control
women who underwent hysterectomy, were assayed for
elastin, LOX and elafin. Results showed that significantly
lower elastin and LOX expression in the cardinal ligament
was found for both premenopausal and postmenopausal
women with POP than in controls (P , 0.05). Among
those with POP, the expression of elastin and LOX in postmenopausal patients were significantly lower than in premenopausal patients (P , 0.05). However, the expression
of elafin was not different between premenopausal and
postmenopausal women with POP (P . 0.05). In conclusion, the lower elastin and LOX expression and higher
elafin expression observed in the cardinal ligaments of
women with POP may have contributed to the development
of this disease.
Po55. A simple on-line screening method
for the rapid discovery of Michael
addition acceptors from natural products
Xiaoyu Zhang and Zhongjun Ma
School of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, China
Michael addition acceptors (such as isothiocyanates,
chalcones, miltirones and alkynols) are the molecules in
which olefins or acetylenes are conjugated to
electron-withdrawing groups. They can undergo Michael
addition reaction with critical nucleophilic amino acids and
cellular glutathione (GSH), and thus regulate many signal
pathways in cells. Currently, bioassay-guided fractionation
of natural product extracts for the discovery of potentially
bioactive Michael addition acceptors is popular. However,
assays based on fractionation require multiple iterations to
isolate active constituents and must be accompanied by
conventional structure elucidation analyses such as NMR
and mass spectrometry. Therefore, these procedures are
labor intensive and low throughput. To enhance the
throughput of these biologically active Michael addition
acceptors discovery assays, we established a simple
LC-MS screening method to investigate constitutes in the
natural product extracts that could form GSH conjugates. It
is quicker and therefore higher in throughput than cellbased assays.
Three kinds of natural products, Echinops grijisii (containing alkynols), Salvia miltiorrhiza (containing miltirones) and Angelica keiskei (containing chalcones), are
chosen for screening assay. LC-MS is employed to analyze
the extract before and after it is incubated with GSH in
vitro. NQO1 induction assay is used to determine whether
the test compound possesses NOQ1-inducing activity, one
of the Michael addition acceptors-mediated activities.
Crystal violet assay is used to determine the cytotoxicity of
the test compound. Western blot analysis is used to
chain reaction-restriction fragment length polymorphism
(PCR-RFLP) analysis. The associations between the polymorphisms and risk of HCC were evaluated by multiple
logistic regression analysis while controlling for confounding factors (including age, sex, status of smoking and
drinking, pack-years of smoking and family history), and,
the P values, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Potential modification of the
effect of the polymorphisms on HCC risk was assessed for
the above confounding factors by the addition of interaction terms in the logistic model and by stratification analyses of subgroups of subjects determined by these factors.
The demographic data and selective characteristics of the
cases and controls showed that there was no significant
difference between HCC patients and control subjects in
terms of age (P ¼ 0.46) or alcohol consumption (P ¼
0.64). More men (P ¼ 0.0055), smokers (P ¼ 0.018) and
HBV carriers (P , 0.001) were presented in the cases than
in controls. In addition, more patients had a family history
of HCC in their first-degree biological relatives compared
with controls (P , 0.001). Genotyping results revealed that
there was no PR þ 331G/A polymorphism in this Chinese
population. The minor allele frequencies of the PR
PROGINS polymorphism were 1.6% and 2.9%, respectively, in cases and controls. The PR PROGINS polymorphism was not significantly associated with HCC risk in
overall subjects, HBV carriers, and non-HBV carriers. The
polymorphisms of PR PROGINS may not play a role in
HCC carcinogenesis in this Chinese population.
Abstracts
Po56. Anti-lung cancer active immunity
induced by FasL, B7-1 genes modified
tumor cells
Shiying Zheng 1*, Dong Jiang 1, Jinfeng Ge 1, and Hong Li 2
1
Department of Cardio-Thoracic Surgery, the First Affiliated Hospital
of Suzhou University, Suzhou, China
2
Department of Geriatrics, the First Affiliated Hospital of Suzhou
Research has revealed that one major reason for the low
responseveness of cytotoxic T lymphocytes (CTLs) to
cancerous cells lies in the loss of interacttion between costimulatory factors CD28 and CD152 and B-7 molecules on
tumor cell surface, which results from the low expression of
the costimulatory factors of B-7 family on the surface of
tumor cells. This precludes CTLs partially activated after recognition of antigens from being fully activated, making it
impossible to generate cytokines with immunological attacking activity against tumor cells and membrane lysis signals
(from the Fas-FasL system) as well. In present study, we constructed the recombinant adenovirus vector (AdV) containing
the human FasL and B7-1 genes (termed FB-11), which was
used to transfect human lung cancer cells. In vitro inducing
tumor-specific CTLs from peripheral blood T lymphocytes
(PBT) and tumorigenicity in mice of the FasL/B7-1 modified
lung cancer cells were observed.
C57BL/6 (H-2b) inbred mice at the age of 6-8 weeks were
bought from the Shanghai Laboratory Animal Research
Center of Chinese Academy of Sciences. SV40 promoter
(PSV40) driven human B7-1 cDNA was presented by Professor
Daru Lu at the Institute of Molecular Genetics, Fudan
University, Shanghai. Human lung cancer cell line A-549 was
provided by the Institute of Cell Biology of Chinese Academy
of Sciences, Shanghai. FasL and B7-1 genes were transfected
into human A-549 cells with adenovirus vectors. The positive
clones were selected by G418. FasL and B7-1 were detected
by flow cytometry and RT-PCR. The abdominal infiltrating
lymphocytes and sensitized spleen cells were obtained from
the mice that were immunized with A-549/FB-11 or wild type
A-549 cells intraperitoneally, and the cytotoxicity of these
CTLs against tumor cells was determined by MTT assay.
Results of flow cytometry and RT-PCR showed that FasL
and B7-1 were highly expressed. FasLþ/B7-1þ A-549 cells
(A-549/FB-11) were inoculated subcutaneously in the dorsal
skin of C57BL/6 mice and then decreased their tumorigenicity
greatly (z ¼ 2.15-46.10; P , 0.01). The A-549/FB-11 cellsensitized mice obtained the protective immune activity
against the rechallenge of wild-type A-549 cells (z ¼
2.06-44.30; P , 0.05). It was showed that the cytotoxicity of
CTLs induced by A-549 /FB-11 cells against A-549 was significantly higher than that of CTLs activated by wild-type
A-549 cells (84.1% + 2.4% vs. 30.5% + 2.3%; P , 0.05).
Results suggest that the FasL and B7-1 can effectively
promote the activity of CTLs against esophageal cancer cells.
Overexpression of FasL can still increase the immunogenicity of the lung cancer cell lines in the presence of overexpressed B7-1 gene, which promotes the development of
antitumor immunity in mice. Overexpression of both tumor
immunity-associated gene B7-1 and apoptosis-inducing gene
FasL in one lung cancer cell line can generate synergistic antitumor activity.
investigate whether the objective protein is activated or
inhibited by the test compound. Transwell migration assay
and tube formation assay are used to investigate whether
the test compound could inhibit tumor angiogenesis,
another Michael addition acceptors-mediated activity.
The extracts of Echinops grijisii, Salvia miltiorrhiza and
Angelica keiskei (1 mg/ml) were incubated with GSH
(2 mM) at 378C for 2 h, respectively and incubation solutions were analyzed by LC-MS. Results indicated that
after incubation, the peak eluted at 105.1 min in the extract
of Echinops grijisii, 80.9 min in the extract of Salvia miltiorrhiza and 46.5 min in the extract of Angelica keiskei
disappeared, suggesting that these three constitutes conjugated with GSH totally and thus might be potential
Michael addition acceptors. According to corresponding
mass spectrum of each disappeared peak, these three constitutes were identified as 2-( penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene,
miltirone
and
isobavachalcone. Subsequent NQO1-induction assay indicated that the three compounds all possessed potent NQO1
inducing activities, with CD values (the concentration
required for 2-fold induction of NQO1) of 2.87 +
0.39 mM, 1.04 + 0.23 mM and 3.58 + 0.62 mM respectively.
Further
investigations
indicated
that
2-( penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene
could activate Keap1-Nrf2 pathway and induce phase 2
enzymes effectively, and miltirone could inhibit tumor
angiogenesis in transwell migration assay and tube formation assay. Therefore, the screened compounds were verified to be bioactive Michael addition acceptors. Compared
with the conventional bioassay-guided extraction and isolation of natural products, this screening method used in
this study is higher in throughput and certainly merited
application in the field of drug discovery from natural products in future.
Abstracts
Po57. Apoptosis-inducing effect of
caspase-3 over-expressed on lung cancer
cell line A-549
Shiying Zheng *, Dong Jiang, and Jinfeng Ge
Department of Cardiothoracic Surgery, the First Affiliated Hospital of
Suzhou University, Suzhou, China
Po58. Effects of Fas ligand on ischemia/
reperfusion injury of cardiomyocytes
Dong Jiang, Jinfeng Ge, Shiying Zheng *, and Jun Zhao
We tested the hypothesis that the shedding of membrane
FasL is a mechanism for downregulating FasL/Fas signaling and that both membrane and soluble FasL are involved
in cardiomyocyte hypoxia/reoxygenation (H/R) injury. We
examined the relative importance of mitochondrial damage
and direct cleavage of the executioner caspases by activated
initiator caspase-8 in the propagation of FasL/Fas signaling
activated by either recombinant membrane FasL or H/R. In
neonatal rat cardiomyocytes maintained under normal
culture conditions, recombinant human soluble FasL
increased caspase-3 activation by two-fold but did not
reduce cell viability. In contrast, infection with a recombinant adenoviral vector expressing the non-cleavable human
FasL (Ad2/ nchFasL) resulted in cardiomyocyte death
that was attenuated by soluble FasL. H/R increased the
mRNA levels of both FasL and Fas and activated
caspases-8, -9 and -3, indicating the activation of FasL/Fas
signaling. Z-IETD-fmk and Z-LEHD-fmk, selective inhibitors for caspases-8 and-9, respectively, abolished caspase-3
activation induced by Ad2/nchFasL or H/R. Z-IETD-fmk
also significantly reduced Ad2/nchFasL- or H/R-induced
Lung carcinoma is one of the most common causes of
malignancy-related death in China. Apoptosis is closely
related to tumor. Although there are many factors involved
in apoptotic program, caspases are shown to play a major
role in the transduction of the apoptotic signal and the
execution of apoptosis in mammalian. In this study, the
eukaryotic expression vector of recombinant caspases-3
was constructed and the apoptosis-inducing effect of its
expression on A-549 cell line was observed.
To investigate the apoptosis-inducing effect of
caspases-3 expressed by constructed eukaryotic vector on
lung cancer cell line A-549, plasmid pcDNA/caspases-3
containing all the cDNA sequences of caspases-3 gene
was used as template to amplify the sequences of small
and large subunits of caspases-3 by PCR. Its products were
separately cloned into the SmaI site of pBluescript KSþ
togenerate both plasmids pBS/SS and pBS/LS. The small
subunit fragment was excised from plasmid pBS/SS with
BamHI and then inserted into the BamHI site of plasmid
pBS/LS preceding that of the large subunit to yield
plasmid pBS/Rev-caspase-3. Rev-caspase-3 cDNA was
excised with KpnI þ XbaI and then subcloned into plasmid
pcDNA3.1(þ) to construct Rev-caspase-3 eukaryotic
expression vector pcDNA/Rev-caspase-3, which was used
to transiently transfect A-549 cell line. Cell count, MTT
assay and electron microscopy were used to confirm the
antiproliferation and apoptosis-inducing effect of
Rev-caspase-3 expression on lung cancer cells. Plasmid
pBS/Rev-caspase-3 and eukaryotic expression vector
pcDNA/Rev-caspase-3 were successfully constructed.
A-549 cells were transiently transfected by either pcDNA/
Rev-caspase-3 or pcDNA3.1(þ) for 24, 48, 72, and 96 h
respectively. Cell growth was measured by cell count and
MTT assay.
In cell count assay, the cell numbers were 1.8 106,
1.55 106, 2.0 106, and 3.1 106 in the experimental
group and 2.5 106, 3.1 106, 4.0 106 and 5.7 106
in the control group at 24, 48, 72 and 96 h respectively.
The growth of A-549 cells was suppressed by
Rev-caspase-3 in a time-dependent manner (P , 0.05).
Results of MTT assay were similar to that of cell count
(P , 0.05). The characteristics of apoptosis such as
chromatin condensation, crescent formation and margination were seen and more obvious with time in the
given-experimental period in the experimental group, but
not easily observed in the control group.The expression of
Rev-caspase-3 by the apoptosis of lung cancer cell line
A-549, which may exhibit a potential way in lung cancer
gene therapy.
In the present study, to test the effect of recombinant
caspases-3 on lung cancer cell, we constructed a eukaryotic expression vector of constitutively active recombinant
caspases-3 and used it to transiently transfect lung cancer
cell line A-549. Results showed that the expressed recombinant caspases-3 could inhibit the growth of A-549 cells
in a time-dependent manner. In addition, the
apoptosis-inducing effect of recombinant caspases-3 on
A-549 cells was also evident. This study demonstrates
the possible use of recombinant caspases-3 in lung
cancer gene therapy. But the effects of growth inhibition
and apoptosis induction conducted by recombinant
caspases-3 on other cell lines of lung cancer or on lung
cancer cell in vivo need to be further investigated.
Abstracts
cardiomyocyte death. H/R potentiated membrane
FasL-induced cell death. Shedding of membrane FasL
downregulates FasL/Fas signaling in cardiomyocytes and
both membrane and soluble FasLs contribute to H/R
injury. Activation of FasL/Fas signaling by either recombinant membrane FasL under normal culture conditions or
H/R causes cardiomyocyte death mainly through the mitochondrial damage/caspase -9 activation pathway.
Po59. Relationship between genetic
susceptibility of non-small cell lung
cancer and TGF-bRI gene
Shiying Zheng *, Jun Zhao, and Dong Jiang
Transforming growth factor-beta receptor type I
(TGF-bRI) gene is a tumor-suppressing gene. The loss of
its biological activity due to the mutation of this gene has
been found in a variety of human tumors. It has been
shown that, during the occurrence of cancers in human, the
cells show the insensitivity to growth inhibition mediated
by TGF-b signal transduction. Despite that numerous evidences demonstrated TGF-bRI acted as a tumorsuppressing gene, the relation between the TGF-bRI and
non-small cell lung cancer (NSCLC) is yet understood
poorly. In present study, the TGF-bRI gene mutation and
its relation to the occurrence of non-small cell cancer was
investigated.
The non-small cell tumor tissues and corresponding cancerous tissues were sample from a total of 53 patients
(including 44 males and 9 females) who had just received
the lung resection operation (the patient group). The tissues
were frozen immediately after surgery, and then stored in
the liquid nitrogen. These patients had not received radiation therapy or chemotherapy before the surgery. Beside
the tumor samples, the blood samples from 89 of adults
without the patient history of cancer were included as negative controls (the control group). Those in control group
came from the same geographic region, and their age range
was similar to that of above cancer patients. The questionnaire showed that 58% and 70% of the subjects in the
patient and control groups were non-smokers, respectively.
The entire coding region of TGF-bRI and flanking intron
sequences from 53 NSCLC tissues were examined for
alterations using Single strand conformation polymorphism
(SSCP) and direct sequencing.
Two experimental evidences were obtamed in present
genetic study. Firstly, The PCR-SSCP assay showed that
Po60. A novel potential anti-angiogenesis
agent, furanodiene
Zhangfeng Zhong 1,2, Xiuping Chen 1, Keyuan Zhou 2,
Tie Wu 2, Liao Cui 2, and Yitao Wang 1 *
1
Institute of Chinese Medical Sciences, University of Macau, Macau,
China
2
Department of Pharmacology, Guangdong Medical College,
Zhanjiang, China
This study was designed to evaluate the antiangiogenetic activities of furanodiene (fur), a natural
product isolated from Ezhu, in vitro. Human umbilical vein
endothelial cells (HUVECs) were used and treated with
different doses of fur with or without vascular endothelial
growth factor (VEGF). The anti-proliferative effect of fur
was measured by XTT assay. Its anti-migration and antiinvasion activities were performed with wound-healing
migration model and three-dimensional cell invasion model
respectively. The effects of fur on HUVECs differentiation
were examined by in vitro tube formation on Matrigel.
Related proteins expression was detected by western blot.
Fur could significantly inhibit the proliferation of HUVECs
the genomic DNA-based RFLP profile of the NSCLC
patients had the two variations compared to the control
group: one was at No. 6 exon, while another at No. 7 exon.
The above genomic DNA samples were then sequenced,
and it was found that the 344th codon changed from AAT
into AAC, while the 406th from TTA into CTA. Secondly,
we also found that a SNP polymorphic site (G/A) located
at the 24th bp in No.7 intron. Accordingly, the 53 patients
in patient group could be assigned into three genotypes: A/
A, G/G and G/A; among them were 18 cases (34%) of the
G/G genotype, 24 (45%) of G/A, and 11 (21%) of A/A. To
clarify the relation between this polymorphism and the
incidence of NSCLC, the PCR-SSCP-based genotyping
assay was performed on the blood samples of the 89
patients in control group. Accordingly, G/G, G/A and A/A
genotypes accounted for 34.8%, 58.4% and 6.7% of the
patients, respectively. According to the case-control analysis, we found that the risk rate of falling ill of A/A genotype patients was three-fold higher than that of G/G
genotype ones (OR ¼ 3.16 95%, and CI ¼1.00 to 9.99).
As the first report, this study showed that TGF-bRI gene
is not a frequent site of spontaneous mutational inactivation
while the detected polymorphism could be a susceptibility
allele that predisposes to carcinogenesis of NSCLC. [This
work was supported by a grant from the General Program
of the National Natural Science Foundation of China (No.
30400533)]
Abstracts
in a dose-dependent fashion but failed to inhibit
VEGF-induced proliferation at low dose. Meanwhile, compared with VEGF induced control, the number of invaded
cells and migrated cells were significantly decreased in fur
treatment groups. Furthermore, fur could dramatically suppress tube formation. The protein expression of p-AKT,
p-ERK, ICAM-1, p-P85 and P85 was significantly inhibited by fur. These studies revealed that fur had potential
effect of anti-angiogenesis through suppressing endothelial
cell growth, invasion, migration and tube formation via
regulation of p-AKT, p-ERK, ICAM-1, p-P85 and P85
expression, which might be one of its important anti-tumor
mechanisms.
Po62. Vascular endothelial growth factor
1498C/T, 936C/T polymorphisms
associated with increased risk of
colorectal adenoma: a Chinese
case-control study
Xianglei Wu 1, Dongqing Li 2, Zhisu Liu 1, and Qun Qian 1*
1
1
Department of Colorectal Surgery, Zhongnan Hospital of Wuhan
University, Wuhan, China
2
Department of Microbiology, School of Basic Medical Science,
Wuhan University, Wuhan, China
*Correspondence address: Tel: þ86-27-67813130;
The tmprss2-ets gene fusions occurred recurrently in
high proportion of human prostate cancer patients in
Western countries. However, the frequency of this type of
gene fusion in oriental population is relatively unknown.
Therefore, in this study, we used nested RT-PCR to characterize the prevalence and diversity of this type of gene
rearrangements in Chinese prostate cancer patient population. Nested PCR was performed and it was shown that
13 out of 27 biopsy samples were positive for tmprss2-ets
fusion. However, sequencing of the 13 positive samples
revealed 6 false positives, suggesting that the nested PCR
method is only moderately specific for the detection of
tmprss2-ets gene fusion. The occurrence of gene fusion in
this Chinese population (7/27, or 26%) is much lower compared with published literatures regarding Western population. Out of the 7 positive biopsy, 6 are the common
tmp-erg fusion type (the exon 1 and exon 2 of tmprss2
fused to the exon 5 of erg). Interestingly, we firstly identified one case with tmprss2-egr1 gene fusion by sequencing, suggesting that other chromosome rearrangement
occurred besides ETS gene family in prostate cancer. The
hybrid transcript was predicted to encode a truncated
1498C/T and 936C/T are two single nucleotide polymorphisms which are associated with colorectal cancer in
VEGF gene. To determine whether such genetic variability
in VEGF contributes to susceptibility of colorectal
adenoma (CRA), a Chinese case-control study was performed in 224 CRA patients and 200 CRA-free controls.
We collected the clinicopathological data and epidemiological risk factors of each sample. Taqman-Probe assay and
ELISA assay were used to analyze genotype and plasma
VEGF levels respectively. The epidemiological investigation presented that male, cigarette smoker, patient who
carry metabolic syndrome or familial antecedent of adenomas were significantly associated with CRA risk. Plasma
VEGF levels of CRA patients were higher than those of
controls (P ¼ 0.003). The analysis of genotype presented
the carriers with 936CT, CT þ TT and 936 2 T allele had
higher risk of CRA compared with controls (CT vs. CC,
OR ¼ 2.00, 95% CI: 1.23-3.25, P ¼ 0.006; CT þ TT vs.
CC, OR ¼ 2.04, 95% CI: 1.28-3.26, P ¼ 0.003; T 2 ,
OR ¼ 1.91, 95% CI: 1.25-2.91, P ¼ 0.003). Both CRA
and control subjects showed no difference in the genotype
of 1498C/T or the allele frequency of C2/T2, but CRA
patients with haplotype 1498T þ 936T presented higher
risk than those with wild-type 1498T þ 936C. Moreover,
clinicopathological data showed patients carrying 936CT þ
TT and 936 2 T allele demonstrated a tendency for villous
adenoma. In conclusion, plasma VEGF levels were evaluated in CRA patients, and the VEGF 936C/T polymorphism and 1498T þ 936T haplotype were found to be
associated with increased CRA susceptibility.
Zhanghui Chen 1,2, Geming Chen 3, Jindan Luo 3,
Weiping Zhao 4, and Jun Yang 1*
2
Zhejiang Academy of Medical Sciences, Hangzhou, China
3
Department of Urology, the First Affiliated Hospital, Zhejiang
University School of Medicine, Hangzhou, China
4
Department of Urology, Sir Run Run Shaw Hospital, Zhejiang
University School of Medicine, Hangzhou, China
Po61. Detection of tmprss2-erg and
tmprss2-egr1 gene fusion in prostate
cancer from Chinese population
EGR1 protein by NCBI ORF finder, which may have
certain function in prostate cancer tumorigenesis. [This
Science Foundation of China (Nos. 30771826, 30872140);
Abstracts
Po63. Effects of anti-EGFR and anti-ERa
conjugated to gold nanoparticles on the
radio-sensitivities of MCF-7 and
MDA-MB-231
Yanjun Zhang, Lihua Zeng, Juan Guo, and Guozhen Guo *
Department of Radiological Medicine, Fourth Military Medical
University, Xi’an, China
Po64. Differential proteomic analysis of
nasopharyngeal carcinoma tissue in
familial nasopharyngeal carcinoma
Shuhua Chen 1, Dsofa Tian 2, Jiping Zhang 1,
Yingchun He 2, Xihua Chen 2, Yangen Lan 1, Fengni Wen 1,
Shaoxiong Zhou 1, Minqi Chen 1, and Zeqi Huang 1
1
The Second People’s Hospital of Foshan, Foshan, China
TCM University of Hunan, Changsha, China
2
Our aim is to compare the difference of proteome among
familial nasopharyngeal carcinoma (NPC), the scattered distributed NPC and normal nasopharyngeal epithelial tissues
to screen for differential proteins, and provide experimental
evidence for elucidating the molecular mechanisms of familial NPC. The proteins of nasopharyngeal tissue from familial
NPC, the scattered distributed NPC and healthy adults were
seperated by two-dimensional gel electrophoresis (2-DE),
respectively. Then gels were stained by blue silver, scanned
by ImageScanner and analyzed with PDQuest software.
Relative abundance of the protein spots among patients of
familial NPC, the scattered distributed NPC and healthy
adults groups were calculated by comparing spots density
volume on the gel. The differentially expressed protein spots
were identified by peptide mass fingerprint (PMF) using
matrix-assisted laser desorption/ionization time of flight
mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE maps of
Nanotechnology may play a novel role by delivering
agent in a targeted fashion to the malignant cells that will
reduce the systemic toxicity of anticancer drug, and
combine drug and radiation effects. Here, we described a
method based on gold nanoparticles (40 nm) conjugated to
monocloncal antibodies (anti-epidermal growth factor
receptor and anti-estrogens receptor a), to investigate their
interaction at the ability to antiproliferative effect on breast
cancer cells (MCF-7 and MDA-MB-231).
The preparation of gold bioconjugates is based on noncovalent binding of the anti-epidermal growth factor receptor
and anti-estrogens receptor a IgG antibodies at their isoelectric point to gold particles. The anti-ER and anti-EGFR
monoclonal antibodies conjugated 40 nm gold nanoparticles,
naked 40 nm gold nanoparticles, and anti-ER and
anti-EGFR monoclonal antibodies were added, respectively,
and incubated 4 h later, the fresh culture were added. After
incubation, the 96-well plates were irradiated by Co60 g ray
deviser. The dose rates were (0, 98.4, 196.59, 393.59) Gy/
min, respectively. The absorbed doses were (0, 1, 2, 4) Gy,
respectively. After radiation, the culture was maintained
96 h. Cell viability was expressed as percentage of controls
(%CT) by MTT assay. MDA-MB-231 cells were seeded in
6-well plate and the DyLightTM 488-conjugated affinipure
goat anti-rabbit IgG conjugated 40 nm gold nanoparticles
and the fluorescence antibody alone were added, respectively, after 2 h of incubation, the culture were moved and
the fresh culture were added, then using Nikon ECLIPSE
TE2000-S and Nikon Digtal Sight DS-U1 observing the
immunofluorescence distribution.
Anti-EGFR monocloncal antibody (100 ng/ml) conjugated moderate concentration 40 nm gold nanoparticles
(2 mM) showed novel antiproliferation on MDA-MB-231
cells at 1 Gy, 2 Gy, 4Gy radiation, contrast to different concentrations of anti-EGFR monocloncal antibody and different concentrations of 40 nm gold nanoparticles. MCF-7 cells
showed resistance at different concentrations of anti-EGFR
monocloncal antibody-conjugated different concentrations of
40 nm gold nanoparticles. Anti-ERa monocloncal antibody
treatment decreased the viability of MCF-7 cells at 2 Gy,
4Gy radiation, but the high concentration of anti-ERa
monocloncal antibody showed low antiproliferative effects.
MCF-7 cells showed resistance at different concentrations of
anti-ERa monocloncal antibody conjugated different concentrations of 40 nm gold nanoparticles at radiation group.
In immunofluorescence images of fluorescence antibody
(DyLightTM 488-conjugated affinipure goat anti-rabbit IgG)
treated MDA-MB-231 cells, 40 nm gold nanoparticles conjugated green fluorescence only appeared at the cell membrane. However, the green fluorescence could be seen in the
cytoplasm and nucleus when cells were treated with
DyLightTM 488-conjugated affinipure goat anti-rabbit IgG.
It is important to note that targeted delivery is not only
dependent on related receptor expression but also on the
mechanism of receptor endocytosis and dynamics. A clear
correlation of EGFR expression with antiproliferative effect
of anti-EGFR monocloncal antibody-conjugated gold nanoparticles is observed in breast cancer cell lines (high in
MDA-MB-231 and low in MCF-7). Whereas antiproliferative effect is low in MCF-7 cell line, although high concentration anti-ER monocloncal antibody conjugated gold
nanoparticles. [This work was supported by a grant from the
International Science and Technology Cooperation Program
of China (No. 2010DFA31900).]
Abstracts
Po65. Research of secretion of cytokines
of lung adenocarcinoma cell line induced
by body fluid of Ascaris lumbricoides
(ABF)
Yanqin Huang, Fang Yuan, Zhifang Dai, Yinying Hu,
Weidong Peng, and Keng Yuan *
Medical Science Institute of Jiangxi Province, Nanchang, China
The aims of this study are to study the influence of body
fluid of Ascaris lumbricoides on the secretion of
interleukin-6 and transforming growth factor b of lung adenocarcinoma cell line (A549) in the following two aspects:
the relationship between the Ascaris lumbricoides (ABF)
concentration and inducing time with the secretion level of
IL-6 and TGF-b; the relationship between the level of the
two cytokines with the apoptosis of A549 cells induced by
ABF. Cells in the logarithmic phase were counted and
inoculated into 96-well plates. ABF at initial concentration
of 3370 mg/ml was diluted mutltiproportionly into 13.5 mg/
ml. MTT was used to test cell vitality. According to the
results of cytotoxicity experiment, ABF with concentrations
of 10, 100 and 300 mg/ml were chosen to induce A549. For
each group, observations were made at 1, 3, 5 and 9 h after
inducement. Enzyme-linked immunosorbent assay (ELISA)
was used to detect the level of IL-6 and TGF-b in the supernatant of each group and HE stain to assess the presence of
apoptosis and calculate the percentage of cells showing
characteristics of apoptosis. The apoptosis of target cells was
confirmed using DNA agarose gel electrophoresis method.
IL-6 level of treatment groups was significantly lower than
that of the control (P , 0.05), and decreased with increase
of ABF. The minimum concentration was 13.72 + 2.67 ng/
ml after 9 h induced by ABF with the concentration 300 mg/
ml. The control was 26.29 + 3.75 ng/ml. On the contrast,
TGF-b level increased with increase of ABF concentrations
(P , 0.05). The highest concentration was 231.75 +
26.2 ng/ml. The control was 142.28 + 35.65 ng/ml. With
HE stain, characteristic morphological changes of apoptosis
were observed in A549 cells induced by ABF. Compared
with the control group, the apoptotic ratios of treated group
were increased (P , 0.05). At 9 h, the rate of apoptosis was
29.12% + 0.82% in 100 mg/ml group. The genomic DNA
from the group showed a typical ladder-like pattern, but
only one fragment was observed with control cells. It
seemed that ABF induced the changes of IL-6 and TGF-b
in dose- and time-dependent manners in a certain degree.
Apoptosis of A549 cells accompanies the changes in
secretion of IL-6 and TGF-b induced by ABF. Within a
certain scope, ABF induced changes in the level of TGF-b
and IL-6 and apoptosis of A549 cells show concentration
and inducing time dependence. It is suggested that the
occurrence of target cell apoptosis may be related to the
changes in the level of TGF-b and IL-6.
Po66. Role of complement C3f peptides in
minimal residual disease assessment of
acute promyelocytic leukemia
Jiuwei Cui 1 , Tingting Liang 1, Wei Li 1, Guanjun Wang 1,
and Xuemin Zhang 2
1
Cancer Center, the First Hospital of Jilin University, Changchun,
China
2
National Center of Biomedical Analysis, Beijing, China
The complete remission (CR) rate of patients with
acute promyelocytic leukemia (APL), who are positive
nasopharyngeal tissue from familial NPC, scattered distributed NPC and healthy adults were acquired. Thirteen differentially expressed protein spots (more than 3 folds) were
identified. In the comparative analysis of the cases of familial NPC in the surveyed pedigrees vs. healthy controls, it
was shown that there were three proteins that downregulated
in their expressive activities, i.e., neuron-specific X11
protein, 70 kDa heat shock protein and activator of 90 kDa
heat shock protein ATPase homologue. One protein upregulated in expressive activity, i.e., peroxiredoxin TSA1, among
the cases of NPC in the surveyed pedigrees, while two proteins were confirmed with no expressive activities, i.e. transferrin receptor protein 1 and putative one, among the
healthy controls. Moreover, the comparing analysis, made
on familial NPC cases of surveyed pedigrees vs. randomly
distributed NPC patients in common population, showed
that there was one protein, intelectin-1, down-regulated in its
expressive activity, and there were four proteins, i.e.,
glucose-6-phosphate 1-dehydrogenase, Rho GDP dissociation inhibitor (GDI) beta, CAP, adenylate
cyclase-associated protein 1 and lactotransferrin, upregulated
in their expressive activities among the former group of
cases. The functions of these proteins involve in heredity
and transcription, metabolism, energy generation, transportation, antioxidation, translation and protein folding etc.
Through comparative proteomic analysis of nasopharyngeal
tissue among familial NPC, scattered distributed NPC and
healthy adults, thirteen differential proteins possibly
involved in familial NPC carcinogenesis were identified.
These data will be helpful to elucidate the molecular mechanisms of familial NPC. [This work was supported by the
grants from the National Natural Science Foundation of
China (30572408, 30672738, and 30973856).]
Abstracts
Po67. Kinetic of plasmid in tissues after
oral administration of recombinant
attenuated Salmonella typhimurium
carrying plasmid pcDNA3s in mice
Xixiu Fang 1,2 *, Dongmei Wang 1, Huangyin Yun 1,
Yuyong Chen 1, Qin Feng 1, Xingang Hu 1, Guowei Le 2,
and Liujian Wen 2
1
Nutrition and Biotechnology Research Center, Jiangsu Animal
Husbandry and Veterinary College, Taizhou, China
2
The Key Lab of Food Science & Safety, Ministry of Education,
Jiangnan University, Wuxi, China
A novel vaccine against hepatitis B virus (HBV) was
designed by putting a naked DNA vaccine carrying hepatitis B surface antigen (HBsAg) into live-attenuated
Salmonella typhimurium. The sensitivity of PCR reaction
was determined by PCR amplification of the control tissue
genome after administering different copies pcDNA3s
plasmid. The plasmid clone in feces bacterial in the intestinal was screened by ampicillin. The results showed that
plasmid DNA was detected in almost all tissues 1, 3, 6, 24,
48 h and 3 weeks after oral administration of pcDNA3
plasmid. Foreign plasmid, however, was detected only in
kidney at sixth weeks. The distribution and kinetics of
plasmid in different tissues was detected by semiquantitative PCR technique after extracting total DNA from
tissues. Genomic DNA was assayed for integrated plasmid
by PCR after purification of high molecular weight
genomic DNA away from free plasmid using gel electrophoresis. The plasmid has been cloned in feces bacterium
in the intestinal. The results showed that foreign plasmid
was detected in thymus and gonads 4 weeks later and
kidney 7 weeks later in mice by the oral. Foreign plasmid
mainly survived as fragments in vivo. Each tissue genome
was negative after PCR amplification and it was lower than
the sensitivity of PCR reaction. No positive plasmid clone
was detected in feces bacterium, suggesting that plasmid
DNA could not be transformed into bacterium in the intestine. In conclusion, foreign plasmid can be absorbed by
gastrointestinal tract and distributed in different tissues
quickly, surviving as the form of fragment in vivo. Foreign
plasmid DNA could not integrate into the host genome
after oral administration.
for PML/RARa fusion gene transcripts and treated with
standard regime of all-trans retinoid acid and/or arsenic
trioxide, is very high (.90%). However, relapse remains
a very important issue for those patients. It is considered
that minimal residual disease (MRD) is the cause of
recurrence, and has been proven to be an independent
prognostic factor for APL. So far, MRD was detected by
RT-PCR for PML/RARa transcript from the bone
marrow sample. There is no serum biomarker available
for APL, although serum is a convenient source of biomarkers. In our previous study, we performed bead fractionation/MALDI-TOF-MS analysis on serum peptidomic
spectrum from patients with acute leukemia and healthy
control. As a result, it was shown a good discriminatory
performance, with 97% sensitivity and 100% specificity.
In this study, we try to investigate the serum biomarkers
for MRD assessment in APL patients with the use of
proteomic
method
based
on
beads
fraction/
MALDI-TOF-MS method and explore the clinical significance of the biomarkers. The sera from 30
patients with APL were analyzed for the peptide spectrum by bead fractionation/MALDI-TOF-MS during the
different stages of the disease, that is, at diagnosis, hematological CR, molecular CR, and the peptide sequencing
was detected by FT-ICR-MS. 5 ml of serum is used for
the fractionation, and MALDI-TOF-MS is sensitive to
detect peptides at sensitivity of a 1fmol concentration.
With the increase of remission degree, two peptide of m/
z 1778.05 and 1865.13 were found to be gradually
decreased in their relative intensities. With FT-ICR-MS
detection, both the peptides were identified as fragments
of complement C3f. Furthermore, compared with the
bone marrow RT-PCR results showing molecular CR in
3 patients with APL, the two serum peptides still had
weak relative intensity, but higher relative intensity than
that
of
healthy
control.
Bead
fractionation/
MALDI-TOF-MS is potentially suitable for monitoring
MRD level in APL patients. It is likely that the intensity
of the two peptides in APL patients with PML/RARa
becoming negative by PT-PCR after treatment could
indicate the existence of residual leukemic cells. Further
investigation with a large samples size and long-term
monitoring is needed to show that peptide features will
be appropriately validate and adapted for MRD assessment in APL patients.
Abstracts
Po68. Cervical cancer SiHa cells inhibited
by nordihydroguaiaretic acid is related to
acetylated histones associated with p21
gene
Peng Gao, Fei Zhai, and Jie Zheng
Department of Pathology and Pathophysiology, School of Medical
Science, Southeast University, Nanjing, China
Po69. Growth arrest of cervical cancer
cells with NDGA is independent of HPV
E6/E7 oncogene expression
Peng Gao, Fei Zhai, and Jie Zheng
Department of Pathology and Pathophysiology, School of Medical
Science, Southeast University, Nanjing, China
Continuous expression of human papillomavirus (HPV)
E6/E7 is required to maintain the transformed malignant
phenotype of cervical cancer cells. Therefore HPV E6/E7
is an ideal target for cervical cancer therapy. Our results
showed that NDGA inhibited the growth of SiHa cells
(HPV16-positive cervical cancer cells) in a dose-dependent
manner, however, HPV16 E6/E7 expression varied with
Po70. Lanthanum inhibits the production
of inflammatory mediators in
LPS-challenged mice via NF-kB pathway
Fei Guo, Yuanlei Lou, Guohui Li, and Yang Wang *
The First Affiliated Hospital of Nanchang University, Nanchang, China
Lanthanide ions have been proved to have various biologic effects. Lanthanum with extremely active physical and
chemical property was evidenced to possess antibacterial
and immune adjustment effects. In the present study, the
effects of lanthanum chloride on lipopolysaccharide (LPS)
challenged mice were examined in vivo and in vitro. Results
indicated that lanthanum chloride can greatly decrease the
secretion of TNF-a and IL-1b as well as TNF-a mRNA
expression in the mice challenged with LPS. To clarify the
mechanism involved, the effects of lanthanum chloride on
the activation of nuclear factor (NF)-kB were examined
both in liver and in peritoneal macrophages. The
LPS-induced activation of NF-kB was significantly blocked
by lanthanum chloride. To verify the relationship between
lanthanum ion and the NF-kB pathway, we detected the
NF-kB/p65-DNA binding activity. At the presence of
2.5 mM LaCl3, DNA binding activity of NF-kB/p65 from
nuclear extract of LPS-induced peritoneal macrophages
decreased significantly. With increasing LaCl3 dosage, the
inhibitory effects progressively increased and the binding
activity was completely abolished at the highest dose
(100 mM). These findings demonstrate that the inhibition of
the LPS-induced inflammatory media, such as TNF-a and
IL-1b, by lanthanum chloride, is due to the inhibition of
NF-kB activation.
Nordihydriguaiaretic acid (NDGA) is a nonspecific
inhibitor of lipoxygenase purified from Larrea divaricata.
In recent years, NDGA has been noted to have potential as
anticancer and antiviral agents. Results reported here
showed that NDGA could significantly inhibit the proliferation of cervical cancer SiHa cells in dose- and timedependent manners. This effect is apoptosis-independent
and associated with G1 arrest, which was verified by flow
cytometry. Further research attributed G1 arrest to upregulation of p21, a critical mediator of G1/S transition.
Western blot and chromatin immunoprecipitation (ChIP)
analysis showed that upregulation of p21 was associated
with histone H3 acetylation, which might be a result of
decreased silencing mediator for retinoid and thyroid
hormone receptors (SMRT) transcription, a member of
histone deacetylase (HDAC) complex. These results
suggest a novel mechanism for NDGA through promoting
histone H3 acetylation to regulate p21 expression, and this
could be, in part, responsible for anticancer effect of
NDGA.
NDGA concentrations in a “V” type in both mRNA and
protein levels, that is, HPV16 E6/E7 expression declined in
0-40 mM NDGA, and recovered in 60-100 mM NDGA.
Further studies indicated that the inhibitory effect of
NDGA on SiHa cells was associated with up-regulation of
three tumor suppressors p21, p27 and p53. Although HPV
E6 or E7 can inhibit the functions of these tumor suppressors, modification of them (such as phosphorylation) can
prevent them form inhibiting. These results demonstrate
that growth of HPV16-positive cancer cells can be arrested
by NDGA despite ongoing transcription of HPV16 E6/E7,
and up-regulation of p21, p27 and p53 may underlie this
effect.
Abstracts
Po71. Determination and clinical
significance of intestinal mucosal barrier
function in children with
hand-foot-mouth disease
Juan Yang 1, Yongkun Huang 1 *, Meifen Wang 2,
Zengqing Du 2, Canlin He 2, Mei Liu 1, Xiaolin Gao 1, and
Lifan Zhou 1
1
Depatment of Pediatrics, the First Affiliated Hospital, Kunming
Medical Colledge, Kunming, China
2
No. 1 Deparment of Medicine, Children Hospital in Kunming,
Kunming, China
Maomin Sun 1, Shiying Zheng 2 *, Hong Li 3, Dong Jiang 2,
and Jinfeng Ge 2
1
Boxi Institute of Clinical Anatomy & Cytoneurobiology Lab,
Medical College of Suzhou University, Suzhou, China
2
Department of Cardiothoracic Surgery, the First Affiliated Hospital
3
This study was designed to assess E-cadherin (E-cad)
and proliferating cell nuclear antigen (PCNA) expression
as well as their clinicopatholoca1 significance in human
non-small cell lung cancers (NSC LCs). Possible molecular
mechanisms of diferentiation and metastasis of NSCLCs
are discussed. Immunohistochemical and immunofluorescence double staining were performed to exam ine the
expression of E-cad and PCNA in 68 primary NSCLCs
cases. The E-cad expression in squamous cell carcinomas
and adenocarcinom showed no significant difference.
E-cad expression had a positive correlation with the
histological-diferentiated grade. A significant difference of
E-cad expression was found between metastatic and nonmetastatic groups. PCNA expression in squamous cell carcinomas and adenocarcinomas showed no significant
difference. The PCNA expression had a reverse correlation
with the histological-differentiated grade. A significant
difference of PCNA expression was found between metastatic and non-metastatic groups. The E-cad and PCNA
expression presented a reverse correlation. E-cad expression
is not associated with the histological type of NSCLC, but
is associated with diferentiation and metastasis of cancer.
Downregulation of E-cad expression affects the proliferation of cancer cells. Conjoint analysis of E-cad and PCNA
expression is a good way to evaluate tumor biologica1
behavior.
Po73. MiR-214, a potential marker for
cell viability
Nan Ye, Shuchun Li, Gaofeng Liang, and
Zhongdang Xiao *
State Key Laboratory of Bioelectronics, School of Biological Science
& Medical Engineering, Southeast University, Nanjing, China
MicroRNAs are small, non-protein coding RNA molecules known to regulate the expression of genes by
Our aim is to explore the change and clinical significance of plasma endotoxin, diamine oxidase (DAO) and
D-lactate in children with hand-foot-mouth disease
(HFMD). Forty children with HFMD according to guide
on the prevention and control of HFMD under Ministry of
Health of China in 2009 were selected as study group in
Children Hospital in Kunming from May to July in 2009.
While 30 healthy children as controls. The study group
was divided critical ill group (n ¼ 14) and no-critical ill
group (n ¼ 26). All cases were required to the blood from
vein. The plasma endotoxin, DAO and D-lactate were
detected by spectrophotography and the differences in all
groups were analyzed. The level of endotoxin in study
group was significantly higher than in control group (t ¼
4.577; P , 0.01), and the level in critical ill group was
higher than that in no-critical ill group and control group
(F ¼ 40.638; P , 0.01). There was no statistic significances of DAO in study group and control group (t ¼
0.978; P . 0.05), but the level in critical ill group was
higher than that in no-critical ill group and control group
(F ¼ 6.399; P , 0.01). The level of D-lactate in study
group was higher than that in control group (t ¼ 3.419;
P , 0.01), and the level in critical ill group was higher
than that in no-critical ill group and control group (F ¼
10.796; P , 0.01). The plasma endotoxin, DAO and
D-lactate can be used as a sensitive marker to early diagnose gastrointestinal mucosal barrier dysfunction. The
damage of intestinal mucosal barrier function was existed
in children with critical HFMD.
Po72. Clinicopathological significance of
E-cadherin and PCNA expression in
hunman non-small cell lung cancer
Abstracts
References
1 Yang H, et al. MicroRNA expression profiling in human ovarian cancer:
miR-214 induces cell survival and cisplatin resistance by targeting PTEN.
Cancer Res 2008, 68: 425– 433
2 Tokumitsu W, et al. Dnm3os, a non-coding RNA, is required for normal
growth and skeletal development in mice. Dev Dynamics 2008, 237:
3738– 3748
3 Barberi-Heyob M, et al. Sequence-dependent growth-inhibitory effects of
the in vitro combination of fluorouracil, cisplatin and dipyridamole. Cancer
Chemother Pharmacol 1993, 33: 163– 170
4 Chen CF, et al. Real-time quantification of microRNAs by stem-loop
RT-PCR. Nucleic Acids Res 2005, 33: e179
Session 3: Stem Cells
Po74. The role of microRNA-1 in
mesenchymal stem cells diffenentiation
into cardiomyocyte-like cells
Tong Wen 1,2, Junyi Zeng 1,2, Yang Wang 3,
Qiongfang Ruan 3, Yuanlei Lou 3, Menghong Wang 1,2,
Guoqiu Ying 1,2, Haiyan Deng 1,2, and Yunfeng Wei 1,2 *
1
Department of Cardiology, the First Affiliated Hospital of Nanchang
University
2
Institute of Hypertension of Jiangxi Province
3
MicroRNAs (miRNAs) are a recently discovered class of
endogenous, small, noncoding RNAs, typically 18-22 nt in
length that mediate post-transcriptional gene repression by
inhibiting protein translation or destabilizing the RNA transcripts of target genes. It is reported that miRNAs regulate
about 30% of encoding genes of the human genome. The
identification of miRNAs expressed in specific cardiac has
led to the discovery of important regulatory roles for these
small RNAs during cardiomyocyte differentiation, cell
cycle, conduction and during stages of cardiac hypertrophy
in adults. Recent findings revealed a muscle-specific
miRNA-1 that plays important roles in heart development
and muscle differentiation. These studies suggest that
miRNA-1 promoted cardiomyocyte progenitor cells and
embryonic stem cells differentiation into cardiomyocytes
via repression of histone deacetylase 4 (HDAC4), Sox6
and the Notch ligand Delta-like 1 (Dll-1). It was reported
that transfection of miRNA-1 in HeLa and C2C12 cells
has been shown to shift the gene expression profile toward
that of muscle cells by targeting HDAC4. Mesenchymal
stem cells (MSCs) from bone marrow are multipotent stem
cells that have the capacities to self-renew and to differentiate into multiple lineages. It has been shown that MSCs
binding to the 30 -UTR region of mRNAs. Several published reports have shown that the expression levels of
some miRNAs are specially regulated during a wide range
of physiological and pathological processes, and miRNAs
can serve as diagnostic biomarkers. miR-214 was reported
to directly bind the PTEN 30 -UTR, leading to inhibition of
PTEN translation and the subsequent activation of the
PI3K/AKT pathway [1]. Also miR-214 was indispensable
for normal skeletal development and body growth in
mammals [2]. To explore the relationship with cell viability, miR-214 was quantified using real-time qPCR in
normal PC12 cells and cells cultured with different serum
concentration. The relative expression level of miR-214
was compared with cell viability results from MTT assays.
Normal PC12 cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and
1% penicillin-streptomycin in a 5% CO2 incubator at 378C.
Cells were planted at the initial density of 2 104 cells/ml
in 35 mm culture dishes or 96-well microtitration plates,
waiting for subsequent treatment. MTT assays were carried
out every one hour after switching to serum-free medium
in batches [3]. The relative expression levels of miR-214
were examined using real-time qPCR with different serum
concentrations at 10%, 4%, 1%, 0.5%, 0.1% or serum-free
medium. Total RNA was extracted using Trizol reagent,
and qPCR was performed with specific stem-loop primers
for miR-214, relative expression was normalized using
small nuclear RNA U6 [4].
In normal culture, the miR-214 expression level was
relatively stable within an entire cell cycle, while combined
with microscopy observation we found it significantly
increased after intensive cell growth with the time. Under a
critical serum concentration of 1%, with lower serum concentration the corresponding changes in cell viability were
immediately related to the relative expression level of
miR-214. miR-214 was significantly upregulated to more
than 3 folds within 2 to 4 h under serum starvation, maintaining a relatively high expression level to induce cell survival in the next two days. However compared with normal
cultured, MTT values of serum-free cultured PC12 cells
maintained almost the same level in the first 12 h, followed
by a rapid decrease to zero thereafter. Cell viability in
serum-free cell starvation is a process of gradual lost, the
cells must have reached a lack of nutrition before succinate
dehydrogenase which was tested in MTT shows significant
reduction.
Two days later, serum-free culture cells were transferred
to normal culture, cells returned to normal growth in one
day, and the corresponding expression level of miR-214
fell back to much the same. Therefore, miR-214 can represent dynamic viability changes in a timely manner, its
expression level could serve as a potential endogenous
marker for cell viability.
Abstracts
showed that the expression of GATA4, NKx2.5 and
MEF2C gradually increased. The immunofluorescence
showed that the cTnI expression was detected at day 4,
6. In untransfected group and transfected empty plasmid
group, the expression of GATA4, NKx2.5, cTnI were not
examined and MEF2C expression had no changes.
miRNA-1 can induce MSCs differentiation into
cardiomyocyte-like cells. It may be a new target for treatment ischemic heart disease with MSCs.
Po75. Mesenchymal stem cells grafting in
infarcted myocardium: serial assessment
with MR imaging and near infrared
optical dual detection
Yefei Li, Yuyu Yao, and Genshan Ma
Department of Cardiology, Zhongda Hospital, Southeast University,
Nanjing, China
The ideal strategy of mesenchymal stem cells delivery
after myocardial infarction (MI) (intramyocardial injection,
or intravenous delivery) has not been clarified. There are
controversial results including implantation efficiency and
function improvement. We utilized MRI and NIR optical
imaging to explore the effect of different stem cell delivery
modes on cell retention time and cardiac function after MI.
MSCs were isolated from the bone marrow of adult SD
rats, and then labeled with superparamagnetic iron oxide
(SPIO) nanoparticle and carbocyanine dye DiD for noninvasive tracking of labeled cells in living animals. 54 SD
rats (weight, 90-100 g) undergoing ligation of left anterior
descending artery were randomized into 3 groups:
Intramyocardial transplantation MSCs group (IM, n ¼ 18),
intravenous transplantation MSCs group (IV, n ¼ 18) and
control MI group (n ¼ 18). Observation intervals were set
for 1 day and 7 days after MI. One hour after completion
of the ligation, the MSCs solution (1.0 106 cells each
rat) was injected into myocardium or infused into tail vein.
After cell-delivery, cellular engraftment was determined at
day1 and day 7 by measuring in vivo the SPIO and optical
signaling in the infarct. MR images were obtained with a
conventional cardiac-gated fast low-angle shot (FLASH)
sequence on 7.0T Bruker magnetic resonance scanner. The
optical imaging experiments were performed using a commercial CRi’s Maestro in vivo molecular imaging systems
which permit coverage in the red, far-red, and near-infrared
spectral regions. Histological sections with SPIO and DiD
were detected by Prussian blue staining and Nikon fluorescent microscope respectively. Cardiac function was
measured by echocardiogram. In IM group, labeled cells
can differentiate into cardiomyocytes and safely repair
heart attack damage after transplantation MSCs in damaged
heart. However, it has also been demonstrated that the efficiency of MSCs differentiation into cardiomyocytes in vivo
is low and the effect of repairing damaged heart is scant.
Therefore, promoting the differentiation of MSCs into
myocardial cells is urgent issues need to address for treatment ischemic heart disease. Cell fate decisions of MSCs
are dictated by activation and repression of lineage-specific
genes. Emerging evidence indicated that miRNAs played a
critical role in this process. But the role of miRNA-1 in
MSCs differentiation into CMs is currently completely
unknown. In this study, a eukaryotic expression vector for
rno-miRNA-1 was constructed successfully and MSCs
were transfected with it. Results showed that overexpression of miR-1 markedly enhanced the expression of cardiac
transcription factors GATA4, NKx2.5, MEF2C. The
cardiac troponin I (cTnI) protein expression was observed
at 4, 6 days after transfection miRNA-1. These suggest that
miRNA-1 can induce MSCs differentiation into
cardiomyocyte-like cells. miRNA-1 may enhance the efficiency of MSCs differentiation into cardiomyocytes and be
a new target for the therapies of ischemic heart disease
with MSCs.
miRNA-1 genomic sequences were amplified by PCR
and cloned into pSD11-U6/Neo/GFP vector, rno-miRNA-1
eukaryotic expression vectors were constructed. MSCs
were isolated from SD rats according to the method
described by Markino with some modification. Cells were
cultured with Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10% FBS and identified by
immunofluorescence for the expression of CD29, CD34,
CD44 and CD45. The fourth passage of MSCs was transfected miRNA-1 plasmid with Lipofectamine2000.
miRNA-1 expression was detected by quantitative real-time
RT-PCR (qRT-PCR); the mRNA expression levels of
cardiac-specific transcription factor GATA4, NKx2.5 and
MEF2C were detected by RT-PCR; cTnI expression was
measured by immunofluorescence.
PCR identification and DNA sequencing showed that
the eukaryotic expression vectors for miRNA-1 constructed
successfully. The fourth passage of MSCs showed a
fibroblast-like morphology and whirlpool-like distribution.
Immunofluorescence showed that 90% of the MSCs were
positive for integrin family member CD29, adhesion molecules CD44, which are two significant markers of MSCs
and negative for hematopoetic precursor cell markers
CD34, leukocyte antigen CD45. Transfection of miRNA-1
in MSCs 48 h later, 20%-30% of MSCs expressed GFP
and qRT-PCR analysis showed that the expression of
miRNA-1 was markedly enhanced. It has been suggested
that the eukaryotic expression vectors can produce mature
miRNA-1 in MSCs effectively. The results of RT-PCR
Abstracts
were visualized in the infracted myocardium as hypointense areas by serial MRI studies from day 1 to day 7. But
at day 1 the intensity by DiD is significantly higher than
day 7 (P , 0.01). On the contrary, the signal of lungs and
spleens with DiD in IV was stronger than IM (P , 0.01).
However, the signal of heart in IV was not observed by
dual-modal trackings at two time points. The results of
labelled cells tracking were consistent with pathological
detections. The left ventricular ejection fraction of IM was
5.6% higher than IV at day 7, but there was no significant
difference between IV and control group. IM injection of
MSCs resulted in increased engraftment within infarcted
heart when compared with IV infusion, and could improve
cardiac function after myocardial infarction. And therefore,
it may provide an advantage over IV infusion in retention
of the stem cells.
Man Li, Yajun Wang, Wenli Mu, Peng Xu, and
Depei Liu *
Institute of Basic Medical Science; Chinese Academy of Medical
Sciences, Beijing, China
*Correspondence address. þ86-10-65105089;
Our aim is to construct the mouse stem cell line stably
integrated with SATB1 gene. The mouse stem cell
line-SNL cell, Lipo2000 transfection reagent were used to
purify SATB1 target protein with neo resistance gene.
Inoculation of SNL cells was processed with MMC as
feeder cells in 6-well plates. When the cells adhere to the
wall, AB1 cells were inoculated to the feeder cells according to normal seeding density. After 24 h of continuous
cultivation, transfect the purified SATB1 target gene with
Lipo2000. Transfection of GFP was used as a contrast
control. Twenty-four hours after transfection, 10% transfection rate was found in GFP-transfected cells, as observed
by fluorescence microscopy. Culture medium containing
500 mg/ml of G418 was then added for sieving. Ten days
later, 30 monoclonals were picked out and cultivation was
continued for 10 generations. Gene and protein expression
levels of exogenous SATB1 were tested by PCR and
western blot analysis respectively. Among the selected 30
monoclonals, the exogenous SATB1 was expressed in nine
of them, as demonstrated by the significantly increased
gene and protein levels in PCR and western blot analysis.
After cultivating for ten generations, expression of SATB1
Po77. Flk1 and flt1 signaling regulate
self-renewal and differentiation of E12
fetal liver hematopoietic stem/progenitor
cells
Shujin Jiang, Feizhe Xiao, and Xiaodong Na *
Department of Pathophysiology, Zhongshan School of Medicine,
SUN Yat-sen University, Guangzhou, China
Developmental signals existing in the microenviroment
direct the self-renewal and differentiation of hematopoietic
stem cells. Expression of vascular endothelial growth factor
(VEGF) and placenta growth factor (PGF) and their receptors flk1 and flt1 is concomitant with the development of
hematopoiesis. Semisolid colony culture, MTT assay, and
flow cytometry were used to investigate the effect of
VEGF and PGF on the self-renewal and differentiation of
E12 fetal liver hematopoietic stem cells in vitro. The yield
of colony forming unit-granulocyte and macrophage
(CFU-GM) was increased by application of VEGF or PGF.
Moreover, VEGF more effectively supported the growth of
CFU-GM than PGF (VEGF: 51 + 5.82, PGF: 39.2 + 7.52;
P , 0.05). Synergistic effect ofVEGF and PGF on
CFU-GM was not observed. SU5416 (1 mM) inhibited the
effects of VEGF or PGF significantly (P , 0.01) by suppressing the phosphorylation of flk1 and flt1. Flow cytometry results indicated application of VEGF or PGF could
increase the percentage of CD34 or Sca-1 positive cells.
VEGF was more effective in supporting the growth of
CD34 positive cells, while PGF was more potent in supporting the self-renewal of Sca-1 positive cells. The selfrenewal of Sca-1 positive cells was enhanced mostly by
combined application of VEGF (VEGF: 2.25%, PGF:
4.67%, VEGFþ PGF: 5.76%). MTT assay and flow cytometry suggested no significant difference between VEGF
and PGF on the number of cultured fetal liver hematopoietic cells. The expression of AKT was detected by Western
blot analysis and the results indicated that AKT was
involved in signal transduction of flk1 and flt1 on the regulation of self-renewal and differentiation of E12 fetal liver
hematopoietic stem/progenitor cells.
Po76. Construction of the mouse stem cell
line AB1 stably integrated with SATB1
gene
genes was still stable. Mouse stem cell line stably integrated with the exogenous SATB1 gene was successfully
constructed. Our study thus laid ground for the further
functional and signal regulation research of SATB1 in stem
cells.
Abstracts
Po78. Modified method of primary
culture and functional text of mouse renal
proximal tubular epithelial cells
Po79. Expression of notch
signaling-related miRNA in hippocampus
of mice after traumatic brain injury
Luping Zang and Xinrong Wu
Changfu Pan 1, Yang Wang 2, Zhucai Kuang 1, Yuanlei lou 2,
Qiongfang Ruan 2, An Xie 1, Faliang Gao 1, Xiaoli Shen 1,
Xianliang Lai 1, Wei Tu 1, and Zhifeng Deng 1 *
General Hospital of Guangzhou Military Area Command of Chinese
PLA, Guangzhou, China
2
Traumatic brain injury induces various kinds of cellular responses that lead to endogenous neural stem cells
proliferation and differentiation, but the regulation mechanism is not well understood. MicroRNAs (miRNAs) are
emerging as important negative regulators of posttranscriptional gene expression and are considered to be
crucial for proper stem cell maintenance, proliferation,
differentiation and apoptosis in the brain. Recent studies
have demonstrated that some specific miRNAs, such as
mir-326 and mir-34a, can negatively regulate notch1 signaling, thereby inhibiting stem cells proliferation and
inducing apoptosis. We presume that mir-326 and
mir-34a might regulate notch1 expression, and then regulate neural stem cells proliferation after traumatic brain
injury. Mice suffered closed head injury were sacrificed
at 4 h, 1 d and 3 d post-injury. We investigated the
expression levels of mir-326, mir-34a and notch1 mRNA
in mice hippocampus at above three time points by realtime PCR. In addition, we also investigated the protein
expression levels of notch1 and nestin (specific marker
for neural stem cells) in mice hippocampus at above
three time points by immunofluorescence and western
blotting. Our results showed thatmir-326 expression level
was decreased apparently at 4 h and 1 d post injury, and
restored to normal level at 3 d post injury; mir-34a
expression level was decreased apparently at all three
time points post injury. In contrast, notch1 mRNA and
protein expression levels were both increased apparently
at all three time points post injury. Moreover, we found
the number of nestin positive cells was also increased
apparently at all three time points post injury. Overall,
our results suggest that traumatic brain injury can inhibit
the expression of mir-326 and mir-34a, causing increase
in the expression of notch1, and then inducing the proliferation of neural stem cells in hippocampus of mice.
Our finding provides a new sight of regulation mechanism of endogenous neural stem cells proliferation in adult
brain after traumatic brain injury. [This work was supported by the grants from the National Key Technology
R&D Program (Grant No. 2008BAI68B06).]
Our aims are to establish a modified method for the
primary culture of mouse renal proximal tubule epithelial
cells, test the urate uptake function, and to establish a
reliable cell-model for relative pharmacological studies. A
single sample study was performed in Department of
Medical Laboratory, General Hospital of Guangzhou
Military Area Command of Chinese PLA from Dec 2009
to Feb. 2010. KM mice at age of 3 5 weeks were supplied by animal experimental center of General Hospital
of Guangzhou Military Area Command of Chinese PLA.
Kidneys were harvested and the cortices were dissected,
minced, and digested using collagenase type I. Fragments
of the tissues was filtered sequentially with 50 and 200
mesh filter. Then the deposition was dissociated by gradient centrifugation with percoll. The isolate was planted in
the cell culture flasks. The bred cells were subcultured
and identified by SP immunocytochemistry. Urate uptake
function was tested by a range of probenecid and benzbromarone concentrations in transport medium contained
1500 mM urate. The anchorage-dependent rate of segments of renal proximal tubules reached 54.5% after
digested using 400 U collagenase type I for 20 min; the
most suitable time for the first change of the culture
medium is 72 h; log phase began from the fifth day and
the cells’ grouth conditions become bad in the tenth day.
In the transport medium contained 1500 mM urate, the
30-min uptake of uric acid was the highest and was taken
as a measure of an initial rate. Different urate uptake
inhibitions were appeared from different concentrations of
the indicate drugs probenecid and benzbromarone. The
inhibitions of benzbromarone were higher than probenecid. The mouse renal proximal tubular epithelial cells cultured by the modified method will yield rich and
homogeneous harvest, and present well function of urate
uptake. The study offers a model in vivo for pharmacological studies.
1
Abstracts
Po80. Effects of human skin-derived
precursor tranplantation on the
regeneration and function recovery of
cerebral ischemic injury in rats
An Xie 1, Zhifeng Deng 2, Yawei Hu 2, Yuanlei Lou 1,
Wei Tu 2, Lei Wu 2, and Yang Wang 1*
1
2
Po81. Stilbene glycosides improved the
proliferation of mesenchymal stem cells
by enhancing SCF secretion in vitro
Jin Zhang 1,#,*, Jin Huang 1,#, Zhiwei Xu 1,*, Fuping Ding 2,
and Zhenquan He 1
1
College of Fundamental Medical Science, Guangzhou University of
Chinese Medicine, Guangzhou, China
2
College of Nursing Science, Guangzhou University of Chinese
Medicine, Guangzhou, China
#
These authors contributed equally to this work.
*These authors are co-corresponding authors. E-mail: zhjin@gzhtcm.
edu.cn
Our aim is to investigate the effect of stilbene glycosides
(SG) in vitro on bone marrow mesenchymal stem cell
(MSC) proliferation and its mechanism. The SG induced
proliferation effect on MSCs was detected by MTT; different cytokine mRNA expression levels were detected by
RT-PCR; SCF mRNA expression was detected by realtime PCR; SCF protein expression was detected by western
blot; SCF content of supernatant was detected by ELISA
assay. Different concentrations (0.1, 0.2 and 0.3 M) of SG
significantly promote the proliferation of the role of MSCs
(P , 0.05), but 0.1 M SG has the most obvious effect
(P , 0.01). 0.1 M SG significantly promoted SCF mRNA
expression, SCF protein expression and SCF secretion
(P , 0.05). SG can promote the proliferation of MSCs,
SCF mRNA expression and SCF secretion. It is suggested
that the role of SG and its mediated serum promoting proliferation of MSCs may be related to the promotion of the
MSCs to express SCF.
There is considerable interest in the use of stem cells
for treatment of the injured or diseased nervous system.
Although embryonic stem cells and fetally derived neural
stem cells are candidate sources of transplantable neural
precursors, their use is associated with both ethical and
clinical issues, such as the requirement for immune suppression. Recently, a neural crest-related precursor cells
were found in neonatal and adult human skin. These skinderived precursors (SKPs) were capable of differentiating
in culture into neural crest-derived cell types, including
cells with characteristics of peripheral neurons and
Schwann cells. They may well provide an accessible, autologous source of neural crest-derived cell types for treatment of the injured or diseased nervous system.
To isolate SKPs, epidermis and dermis were dissociated
from skin samples, and dissociated dermal cells were cultured with the formation of spheres in suspension by 2
weeks. Then the SKPs adhered and be successfully
expanded as monolayer with fibroblast-like shape. In order
to determine whether SKPs have the effects of promoting
the regeneration and function recovery of ischemic brain
injury, rat middle cerebral artery occlusion (MCAO)
models were produced by obstructing MCA using a single
strand nylon thread, and CM-DiI labeled SKPs were transplanted into rats through the femoral vein. Neurological
function was evaluated with beam-walking, bar-grasping
and water maze. To investigate the survival and differentiation of skin-derived precursors transplanted into rat
brain, TUNEL kits were used to detect the neural cell
apoptosis, and immunofluorescence assay was used to
detect neural associated antigen expression.
Through combination of neural stem cell and fibroblast
culture, SKPs still expressed Nestin and CD29 instead of
CD34 as shown by immunofluorescent staining. RT-PCR
analysis showed that SKPs expressed nestin, p75NTR,
fibronectin, vimentin, Slug and Snail. In vitro, SKPs could
be differentiated into neural-like cells expressing neural
phenotypes by neuron and gliocyte derived signals. Rats
receiving SKPs had a more rapid recovery as evaluated by
beam-walking, bar-grasping and water maze performance
than ischemia control rats. Twenty-four hours later, the
transplanted SKPs were detected mainly in the ischemic
zone. These suggested that the transplanted SKPs can
migrate to the ischemia zone through the femoral vein
graft in a short time. The immunofluorescent staining
results confirmed a part of CM-DiI positive cells
expressed NSE and GFAP also. These data suggested that
some transplanted SKPs differentiated into neural cells.
TUNEL assay showed the number of apoptotic cells in
SKPs transplanted rats decreased significantly compared
with ischemia control rats in the penumbra of cortex with
light microscopic analysis.
In summary, transplanted SKPs can survive in the brain
of MCAO rats and were mainly distributed in the region
around the ischemic focus and remained the differentiation
characters of neural cells. The results demonstrate that
SKPs can improve the neurological function following
focal cerebral ischemia in rats. SKPs is one of suitable
seed cells for treatment of ischemic brain injury.
Abstracts
Po82. In vitro isolation of breast cancer
stem cells by magnetic activated cell
sorting (MACS)
Yingxin Chen, Molin Li, Lianhong Li *, Jun Mao,
Lihui Wang, Yajun Tao, Lixia Wang, and Bo Wang
Basic Medical Science of Dalian Medical University, Dalian, China
1 Ponti D, et al. Isolation and in vitro propagation of tumorigenic breast
cancer cells with stem/progenitor cell properties. Cancer Res 2005, 65:
5506– 5511
Po83. Induction and culture of neural
stem cells derived from human induced
pluripotent stem cells
Nianhua Feng, An Xie, Yuanlei Lou, Qiongfang Ruan,
Fei Guo, and Yang Wang *
Institute of Urology, the first affiliated hospital of Nanchang
University, Nanchang, China
The incidence of serious brain injury and regressive
diseases of central nervous system is increasing and
become one of the most intractable diseases in clinic.
Neural stem cell (NSC) based transplantation is a hopeful
therapy method. However, the isolation of NSCs is difficult and limited NSCs can be expanded in large-scale in
vitro, so there are not enough NSCs for clinic application. Additionally, previous reports indicate that NSCs
in different locations have different properties. Therefore,
it is necessary to find a new suitable source of NSCs.
Induced pluripotent stem cell (iPS cell) is a kind of pluripotent stem cell which can be generated from adult cells.
iPS cells are multipotent and can be propagated in vitro.
It is one of the most promising sources of NSCs for
NSCs-based therapy. However, there are two steps before
its application: Induction and amplification. First, it is
important to establish a stable differentiation system of
iPS cells to neural stem cells; then, a suitable culture
system of iPS cell derived NSCs is also necessary for
long-term amplification. Here, NSCs derived from iPS
cells were obtained through three steps and a long term
culture system was established. First step: iPS clones
removed from feeder layer were resuspended with embryoid bodies (EBs) medium and then cultured for 4 days in
a petri dish to form EBs; Second step: EBs were collected and subjected to a 4 days retinoic acid (RA) inducing process; Third step: after 4 days RA induction, EBs
were transferred to serum free medium for the screening
of NSCs. There were many kinds of growth factors in
serum free medium, which can promote the survival and
propagation of NSCs. EBs maintained in EB medium
were set as control. After 7 days screening, EBs were
collected, named iPS cell derived NSCs. These iPS cells
derived NSCs were maintained in serum free medium
and passaged using mechanical method. Real-time PCR
and immunostaining were used to detect the expression
Breast cancer is one of the most common malignant
tumors diagnosed in female. It is reported that more than a
million women are diagnosed every year all over the
world. The main treatments for breast cancer are surgery,
chemotherapy and radiation therapy. Nearly 9 out of 10 of
women diagnosed with stage I breast cancers survive their
disease beyond five years, while this drops to around 1 out
of 10 diagnosed with stage IV. It is found there are some
“mammosphere-forming” breast cancer cells in tumor
tissues which have stem cell properties and are resistant to
conventional chemotherapy. These cancer stem cells can be
identified by CD44þ/CD242/low [1] and activated some
signaling pathways, such as Notch, PI3K and Hedgehog,
etc. The aim of this study is to find a suitable method for
isolating breast cancer stem cells in vitro. MCF-7 cells in
suspension were cultured in DMEM-F12 with bFGF, EGF,
insulin and B27 to generate primary mammospheres.
Seven days later, 2 107 MCF-7 mammosphere cells were
used to collect CD44þ/CD242 cells by MACS. Reverse
transcription polymerase chain reaction (RT-PCR) method
was used for analyzing the expression of GLi-1
(glioma-associated oncogene homoglog-1), SMOH
(smoothened homolog), SHH (sonic Hedgehog) and PTCH
(human patched gene) in CD44þ/CD242/low and
non-CD44þ/CD242/low cells. The same number of CD44þ/
CD242/low or non-CD44þ/CD242/low cells was inoculated
subcutaneously into Balb/c nude mice respectively. Tumor
growth was assessed by observing the presence of tumor
nodules and measuring tumor volume with a sliding
caliper.
The ratio of CD44 þ/CD242/low subpopulation in
MCF-7 mammosphere cells was (8.90 + 1.73)%. The
expressions of GIi-1, SHH and SMOH in CD44þ/ CD242/
low MCF-7 mammosphere cells are significantly higher
than that in non-CD44þ/CD242/low MCF-7 mammosphere
cells (P , 0.01, 0.05, 0.05 respectively). Tumor growth in
nude mice inoculated with non-CD44þ/CD242/low cells is
faster than those with CD44þ/CD242/low cells.
By using magnetic activated cell sorting (MACS),
cancer stem cells with CD44þ/CD242/low were successfully isolated from MCF-7 mammosphere cancer cells, in
which hedgehog signaling pathway was significantly activated. CD44 þ/CD242/low tumor cells grew slowly in vivo.
Reference
Abstracts
Session 4: Antibody and Medicine
Po84. Efficacy of a bioartificial liver
based on fluidized bed bioreactor for the
treatment of fulminant hepatic failure in
pigs
Guoliang Lv 1,#, Lifu Zhao 1,#, Anye Zhang 1,
Xiaopeng Yu 1, Yingfeng Lu 1, Yu Chen 1, Chengbo Yu 1,
Xiaoping Pan 1, Yimin Zhang 1, Ying Yang 1, Tao Song 2,
Jiangsheng Xu 2, Yu Chen 3, Yuemei Chen 1, and
Lanjuan Li 1 *
1
State Key Laboratory for Diagnosis and Treatment of Infectious
Diseases, First Affiliated Hospital, School of Medicine, Zhejiang
University, Hangzhou, China
2
Institute of Electrical Engineering, Chinese Academy of Sciences,
Beijing, China
3
Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, China
#
We describe a new bioartificial liver (BAL) system
based on a choanoid fluidized bed bioreactor containing
alginate-chitosan encapsulated primary porcine hepatocytes. The feasibility, safety and efficiency of this device
were estimated using a pig fulminant hepatic failure (FHF)
model.
FHF was induced with intravenous administration of
D-galactosamine. Fifteen FHF pigs were equally divided
into three groups: (i) an FHF group which was only given
intensive care; (ii) a sham BAL group which was treated
with the BAL system with empty encapsulation and (iii) a
BAL group which was treated with the BAL system containing encapsulated freshly isolated primary porcine hepatocytes. Biochemical parameters of these animals were
measured, and properties of encapsulation and hepatocytes
post-perfusion were evaluated.
Compared with the two control groups, the BAL treated
group had prolonged the survival time and decreased the
blood ammonia and lactate levels. Blood glucose and
amino acid balance remained stable. No obvious ruptured
beads or statistical decline in viability or function of hepatocytes were observed.
This new fluidized bed bioartificial liver system is safe
and efficient. It may represent a feasible alternative in the
treatment of liver failure.
of neural stem cell marker nestin in primary and subcultured iPS cell derived NSCs, nestin positive cell rate was
calculated. For terminal differentiation, primary and subcultured iPS cell derived NSCs were cultured in DMEM/
F12 contained 10% fetal bovine serum. For clonal analysis, subcultured iPS cell derived neurospheres were dissociated and serially diluted to yield 1-2 cells/10 ml cell
suspension, a 10-ml cell suspension was added to each
well of 96-well plates containing 300 ml serum free
medium. Wells containing one viable cell were marked
the next day, clone formation potency and cell proliferate
were evaluated. On the first day of the third step, EBs
adhered to the bottom of culture dish, and rosette constructions were observed on the 4th day. There were no
rosette constructions in control EBs. Then most EBs were
detached from the bottom and form uniform neurospherelike clones, all clones had clear and bright boundaries,
and continually increased in size. The immunostaining
results showed that, nestin positive cell rate was
87.54% + 3.67% in induced EBs, and 23.77% + 2.96%
in control EBs, there was significant diversity (P , 0.05).
When cultured in serum containing medium, these iPS
cell derived NSC clones differentiated to neurons and
astrocytes which expressed b-tubulin Z and GFAP
respectively. Additionally, real-time PCR indicated that
the expression level of nestin gene in induced EBs were
also significantly higher than control EBs (P , 0.05), the
fold changes was 7.14% + 1.96%. When maintained in
serum free medium, cells inside the clone propagated
fast. After several subcultures, these clones still expressed
nestin, and had terminal differentiation potency.
Moreover, there were 12.5% single iPS cells derived
NSCs survived and formed neurospheres after one week.
Furthermore, iPS cell derived NSCs can be cryopreserved, and proliferated well when thawed. Our results
suggest that three steps inducing method is an effective
mode for the differentiation of iPS cells to neural stem
cells. Serum free medium used in our experiment is suitable for the long-term propagation of NSCs derived from
iPS cells. [This work was supported by the grants from
the National Natural Science Foundation of China (No.
30960385) and the National Key Technology R&D
Program (No. 2008BAI68B06).]
Abstracts
Po85. Aptamer against IL-17RA from
CELL-SELEX as a new adjunctive
therapeutic reagent delayed inflammatory
processes with the inhibition of IL-6 in
murine experimental osteoarthritis
Dongqing Li 1 *, Liang Chen 2, and Xianglei Wu 3
1
Department of Microbiology, School of Basic Medical Science,
Wuhan University, Wuhan, China
2
Department of Orthopedic Surgery, Renmin Hospital, Wuhan
University, Wuhan, China
3
Department of E.N.T, Zhongnan Hospital, Wuhan University,
Wuhan, China
Po86. The preparation and
characterization of specific monoclonal
antibodies against heavy metals MAbs
that recognize chelated mercury ions
Li Zhao 1,2, Fenglong Wang 1, Hui Yang 2, Peng Li 2, and
Xia Li 2,3 *
Po87. Protamine sulphate-stabilized
calcium phosphate nanoparticle for gene
delivery
Yachun Liu 1,2,#, Qian Liu 1,#, Fangli He 1,#, Tao Wang 1,
Shuanglin Xiang 1*, Shengpei Su 2, and Jian Zhang 1 *
1
Key Laboratory of Protein Chemistry and Developmental Biology,
Ministry of Education of China, College of Life Sciences, Hunan
Normal University, Changsha, China
2
Key Laboratory of Sustainable Resources Processing and Advanced
Materials of Hunan Province, College of Chemistry and Chemical
Engineering, Hunan Normal University, Changsha, China
#
*Correspondence address. E-mail: [email protected] (S. X);
[email protected] (J. Z.)
1
College of Animal Science and Medicine, Inner Mongolia
Agricultural University, Hohhot, China
2
Vegetable Research Center of Beijing Academy of Agriculture and
Forestry Sciences, Beijing, China
3
Beijing Research Center for Agri-food Testing and Farmland
Monitoring, Beijing, China
The environmental pollution by heavy metals such as
mercury, cadmium and lead has become a worldwide public
Calcium phosphate (CaP) is one of the mostly used nonviral vectors for in vitro transfection in a variety of mammalian cells due to its low toxicity, biodegradability and
ease of use. However, the transfection efficiency of the
classical CaP method strongly depends on the preparation
parameters, such as pH value, concentrations of CaCl2 and
DNA, temperature, the time between precipitation and
transfection, and cell types. The reproducibility of this
method is poor, and its transfection efficiency is relatively
Osteoarthritis (OA) is characterized by synovial inflammation and progressive cartilage degradation. Cytokine IL-17
family and one of its receptors IL-17RA are deeply associated
with OA pathogenesis. IL-17RA mediated signals transduction is required to the expression of critical genes which are
contributed to inflammation during OA pathogenesis. In this
study, we applied a cell-SELEX method to obtain a single
aptamer (RA10-6) that can binds to IL17RA with high affinity
and specificity. The whole selection processes were based on
two types of NIH3T3 cells which have the marked difference
on IL-17RA expression level. We found RA10-6 could efficiently block IL-17 binding to IL-17RA in vitro. In the
murine experimental osteoarthritis model, RA10-6 can down
regulate the synthesis of IL-6 in synovial tissues especially
synergized with celexib. Our results present an initial study
that RA10-6 has inhibitory effects on IL-17/IL-17RA signals
in OA, and it may be used as a potent adjunctive therapeutic
agent for the early treatment of osteoarthritis.
health hazard. To rapidly and inexpensively monitor
environmental heavy metals is a prerequisite for minimizing
human and animal exposure. The development of immunoassays to detect mercury ion residues is a promising strategy with the advantage of rapidness and cheapness. We
reported the isolation and characterization of mercuryspecific monoclonal antibodies. Since Hg2þ ions are too
small to elicit an immune response, the metal was coupled
to protein carrier (keyhole limpet, KLH) using a chelator
(diethylenetriamine pentaacetic acid, DTPA). After the successful synthesis of antigen and characterization, monoclonal antibodies against mercury ions were generated by
immunizing BALB/c mice with mercury conjugated antigen
(Hg-DTPA-KLH). The stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma
cells. The hybridoma cells were subcloned by the limiting
dilution and screened by ELISA, two hybridoma cell lines
producing stably specific monoclonal antibodies (MAbs)
against mercury ions were obtained, named H2H5 and
H1H8. The ascites fluid was produced in BABL/c mice by
intraperitoneal injection of 1 107 H2H5 and H1H8 cells,
respectively. The titers of ascites were all above 1:51200.
The isotyping of secrete antibodies from two hybridoma cell
lines was IgG1, kappa type. These data laid a potency of
establishing immunoassays methods of determining Hg2þ
ion residues and had the realistic significance for improving
the efficiency and quality of risk assessment.
Abstracts
Po88. Experimental analysis of human
body fluid proteomics (HBFP) in the
pregnancy disease research
Yanling Liu and Songping Luo
Deptartment of Gynecology, the First Affiliated Hospital, Guangzhou
University of TCM, Guangzhou, China
Human body fluid contains proteins that can be informative for disease detection and surveillance of human health.
Comprehensive analysis and identification of the proteomic
content in human body fluid is a necessary first step
toward the discovery of human body fluid protein markers
for human disease detection. The article will review the
recent advances in human body fluid proteome analysis in
the reproductive research, including serum/plasma,
cervical-vaginal fluid, follicular fluid, amniotic fluid, cord
blood, as well as its applications to pregnancy disease
research. And discuss some of the critical challenges and
perspectives for this emerging field.
Po89. Express PD-L1 fusion protein by
Prokaryotic system and PD-L1
monoclonal antibody preparation
Liping Shen, Jiandong Li, Long Xu, Tao Bian,
Siyong Chen, Feng Qiu, and Shengli Bi
National Institute for Viral Disease Control and Prevention, Center for
Disease Control and Prevention, Beijing, China
Programmed death 1(PD-1)/programmed death 1 ligand
1 (PD-L1) signal passway is highly relevant with immune
tolerance. Blocking the passway could restore the function
of exhausted T cells; improve the ability of virus clearance.
In the study we express and purify the PD-L1 fusion
protein and prepare the anti-PD-L1 monoclonal antibody
for further work in which we would study the possibility
and method about immunity therapy by blocking PD-1/
PD-L1 passway with antibody medicine.
Using codon optimized and synthesized the whole gene
sequence of PD-L1 that obtained from GenBank, we constructed the expression plasmid of PD-L1 and expressed the
fusion protein in E. coli. After obtaining the PD-L1 protein,
we conjugated the HRP on the PD-1, coated PD-L1 fusion
protein on 96-well plates and carried out ELISA direct
combine assay. At the same time, Biomolecular interaction
analysis (BIA) assay was also used to analyze the bioactivity of PD-L1 fusion protein. Then we prepare PD-L1 monoclonal antibody by hybridoma technique.
We constructed PD-L1 fusion expression plasmid and
obtained the PD-L1 protein, whose molecular weight of
protein is 47 kDa, purify is 85%, and density is 0.4 mg/ml.
Results of ELISA and BIAcore assay showed that PD-L1
fusion protein can combine with PD-1 specially. Identified
by competitive inhibition ELISA and western blotting
analysis, 5 cell strains of anti-PD-L1monoclonal antibody
were selected successfully.
We have obtained PD-L1 recombination protein and
anti-PD-L1 monoclonal antibody cell stains successfully.
Results could provide basic conditions for further antibody
medicine study and engineering antibody work.
low. It is now known that the particle size of CaP affects in
a great degree the transfection efficiency. So far, many
efforts have been afforded to prepare the CaP particles with
appropriate size including the creation of CaP nanoparticles
to facilitate delivery of DNA into cells through endocytosis.
In this study, we have developed a protamine sulphate
(PS)-modified calcium phosphate nanoparticle formulation
(PSCaP) for efficient delivery of DNA into cells, based on
that the adsorption of protamine sulphate onto surface of the
CaP particles could decrease and stabilize its size.
To evaluate whether protamine sulphate modified CaP
could stabilize the particles size and thus enhance the transfection efficiency, pEGFP-C1 green fluorescent protein was used
as an indicator of transfection efficiency, atomic force microscope was used to evaluate the morphological change and the
size of the CaP particles, and MTT assay was introduced to
detect the cell viability and inhibition effect. The classical CaP
method was used as control. In 293 FT, HEK 293 and NIH
3T3 cells, the transfection efficiency of the PSCaP was
obviously higher than classical CaP method. In addition, the
PSCaP was more stable and could be used for transfection
after one week of storage at 48C without loss of efficiency,
indicating that PS may increase the stability of the CaP nanoparticle. Atomic force microscope image also showed that the
size of the PSCaP (Ca.30 nm) was much smaller than the classical CaP particle (Ca.150 nm). This implies that the smaller
nanoparticle could improve the transfection efficiency, and
might promote delivery of DNA into cells through endocytosis. Cell viability inhibition assay indicated that both methods
showed same cell toxicity. In conclusion, the PSCaP can be
used as a better non-viral transfection vector in comparison
with the classical CaP vector. [This work was supported by
the grants of 863 Project of Ministry of Science and
Technique of China (No. 2006CB943506), the Science
Research Project of department of education of hunan province (No. 09C621), the Postdoctoral Science Research Fund
of department of Science and Technique of hunan province
(No. 2009RS3015), Key Laboratory of Chemical Biology and
Traditional Chinese Medicine Research (Ministry of
Education of china), Hunan Normal University (No.
KLCBTCMR2009-12).]
Abstracts
Po90. Anti-human B7-1 antibody alleviate
mouse lupus-like disease induced by
pristane
Qin Shi 1, Zengyan Gao 2, Fang Xie 3, Yong-ping Gu 3,
Tianjie Yang 2, Li Huang 2, Qihong Qian 1,
and Yuhua Qiu 2*
1
The First Affiliated Hospital of Soochow University, Suzhou, China
Deptartment of Immunology, Medical College, Soochow University,
Suzhou, China
3
Deptartment of Pathology, Medical College, Soochow University,
Suzhou, China
making ones. The control mice’ kidneys had no significant
pathological changes and IC could not be detected.
Mouse anti-human B7-1 antibody 4E5 alleviate mouse
lupus-like disease induced by pristane.The data in vivo
suggest blockade of B7-1/CD28 co-stimulatory signal
pathway is a promising strategy to prevent progression of
lupus-like disease and other autoimmune disease by suppressing T cell-mediated immunity.
2
Po91. The experimental study of lung
carcinoma vaccine modified by human
B7-1 and IFN-g genes
Shiying Zheng 1*, Dong Jiang 1, Jinfeng Ge 1, and Hong Li 2
1
Department of Cardiothoracic Surgery, the First Affiliated Hospital
2
CD80 (B7-1) and interferon-gamma (IFN-g) play important roles in antitumor immunity induced by T lymphocyte.
To study the antitumor immune effects of lung carcinoma
vaccine modified with human B7-1 and IFN-g genes, we
examined the characteristics of a clonally derived A-549
cell line modified with B7-1 and IFN-g genes, and detected
decreased tumorigenicity following their implantation
intrader- mally into human immune reconstituted SCID
mice (hu-PBL-SCID).
Recombinant retrovirus vectors pLXSN/B7-1, pLXSN/
IFN-g, and pLXSN/B7-1þ IFN-g were constructed. In
vitro, the primary lung carcinoma cells A-549 were transfected with the retroviral vector and prepared as a vaccine.
Clonal derivates of A-549 cells were isolated by G418
selection, expanded to cell lines, and named as A-549/
pLXSN, A-549/B7-1, A-549/IFN-g, and A-549/B7-1 þ
IFN-g. The phenotype changes of transduced A-549 cells
were detected by the following methods: immunoflow
cytometry; reverse transcription-polymerase chain reaction
(RT-PCR) and dot blot for the expression and
co-expression of B7-1 and IFN-g gene; flow cytometry for
the individual phases of cell cycles; and cell counts for
growth curves. Then autologous lymphocytes were irritated
with the lung carcinoma cells for competition inhibitory
cytotoxic testing.
A-549 cells were transfected with four kinds of vectors,
expanded to cells and named as A-549/pLXSN, A-549/
B7-1, A-549/IFN-g, and A-549/B7-1 þ IFN-g. Results of
immunoflow cytometry showed that the expression rate of
the CD80 molecule in A-549/B7-1 was 94% and that of
IFN-g in A-549/ IFN-g was 32%, while the co-expression
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by auto-antibodies to diverse
self antigens, immune complex and inflammatory lesion in
many organs. It’s well reported that B7/CD28
co-stimulatory signal plays a crucial role in the pathogenesis of SLE. On the base of mouse anti-human
B7-1antibody (clone 4E5) developed in our lab, we
explored the therapeutic effect of 4E5 on lupus-like disease
induced by Pristane in C57BL/J6 mice.
Forty-five 8-week-old female C57BL/J6 mice were
divided into three groups randomly. The control group was
injected with 0.5 ml 0.9% N.S. while the other two groups
were treated with 0.5 ml pristine to induce lupus-like
disease. One group with pristane was intervened with mouse
anti-human B7-1 antibody by caudal vein injection (the
intervention group) and the other one with mouse isotype
IgG (the model-making group). The activation of macrophage and dendritic cell in local lymph nodes and spleens
and markers on B cells in spleens were measured by FCM,
respectively. Antinuclear antibody (ANA) in serum and proteinuria were detected monthly after injection. 7 months
after injection, all mice were killed and kidneys were slided
and stained with H&E or FITC-labeled IgG to observe the
evidence of glomerulonephritis histopathologically. After
10 days, compared with the control group, macrophage and
dendritic cells in local lymph nodes and spleens from the
intervention group and the model-making group had different levels of activation: the intervention group activation
was significantly lower than the model-making group (P ,
0.05). The expressions of CD21, CD40 and CD86 on spleen
B cell of the intervention group increased lower than those
of the model group (P , 0.05). The concentration of ANA
in mice sera and protein in uria showed the same trend.
Seven months later, the intervention group showed less histopathological damages than the model-making group.
Immunofluorescence intensity of immune complex(IC) in
the intervention group mice was weaker than the model-
Abstracts
Po92. A study of outcomes following
intra-fascial hysterectomy with intact
ascending uterine artery
Xiaoqing Dou 1,3, Yunhai Yu 2,3, and Shiqian Zhang 3 *
1
Department of Gynaecology and Obstetrics, Zhejiang Hospital of
Traditional Chinese Medicine, Hangzhou, China
2
Department of Gynaecology and Obstetrics, the Second Hospital of
Shandong University, Ji’nan, China
3
The State-Key Discipline of Obstetrics and Gynecology, Department
of Gynaecology and Obstetrics, Qilu Hospital of Shandong
University, Ji’nan, China
Our aim is to study the clinical value and outcomes of
intra-fascial hysterectomy (IFH) with intact ascending
uterine artery (AUA). Eighty-four patients were randomly
divided into a control group, who received IH only, and
study group, who received IFH with intact AUA. Various
operative indexes, ovarian function, sexual life, and urinary
and fecal discharge were observed in each group. There
was no significant difference in the various indexes
between the two groups (P . 0.05). Compared to preoperative levels of FSH and E2, postoperative blood levels of
FSH and E2 were significantly different in the control
group (P , 0.05), but not in the study group (P . 0.05). A
significant difference (P , 0.05) in the incident rate of
menopausal symptoms was observed between the two
groups. Sexual life, levels of blood glucose and lipids, and
urinary and fecal discharge after operation did not show
obvious change. The detection rate of bilateral AUA and
ovarian branch of uterine artery (UOA) was 100% in the
study group post-operation. In a short-term observation,
IFH with AUA had little effect on ovarian function, levels
of blood glucose and lipids, or urinary and fecal discharge.
The quality of sexual life of patients received IFH with
AUA was improved than before operation. The blood circulation of AUA and UOA was also not affected after operation. The long-term clinical effects of such hysterectomy
should be further studied.
Po93. Liver-specific expression of an
exogenous gene controlled by human
apolipoprotein A-I promoter
Yurong Hu 1, Xueling Ren 1, Hui Wang 1, Yue Ma 1,
Lei Wang 1, Yingying Shen 1, Kazuhiro Oka 2,
Zhenzhong Zhang 1*, and Yun Zhang 1 *
1
School of Pharmacy, Zhengzhou University, 100 Science Road,
Zhengzhou 450001, China
2
Department of Molecular and Cellular Biology, Baylor College of
Medicine, One Baylor Plaza, Huston, TX 77030, USA
*Correspondence address. Tel: þ86-371-67781910; Fax:
þ86-371-67781907; E-mail: [email protected] (Z.Z);
[email protected] (Y. Z.)
Liver-specific gene therapy is advantageous to minimize
the possible adverse effects caused by non-target gene
expression. The CMV promoter of the enhanced green fluorescent protein (EGFP) expressing plasmid CMV-EGFP
was replaced with the liver-specific promoter apolipoprotein A-I (ApoAI) generating ApoAI-EGFP plasmid. In
vitro expression experiments performed in various cell
lines including HepG2, SMMC-7721, MCF-7, ACC-2 and
Lo2 indicated that pCMV-EGFP treatment caused gene
expression in all the cell lines, whereas pApoAI-EGFP
treatment only induced EGFP expression in cells of liver
origin including the liver cancer cells HepG2 and
rate of the CD80 and IFN-g molecules in A-549/B7-1 þ
IFN-g was 32.2%. There was no expression of the CD80
and/or IFN-g molecule in A-549/pLXSN and parental
A-549. Three to five days after transfection with B7-1 and
IFN-g, the expression rate of the CD80 molecule in the
primary lung carcinoma vaccines was 30%, and that of the
IFN-g molecule was 10%-15%. Competition inhibitory
testing by the MTT method was used to detect the presence
of autologous lymphocyte mediated specific immunity
toward primary lung carcinoma cells. Results showed that
the cytotoxic activities induced by B7-1- and
IFN-g-modified vaccines were much higher than those
induced by single gene-modified vaccines, which were also
significantly higher than those induced by vaccines transfected with the control pLXSN vector. Our findings indicated that lung carcinoma vaccines modified with B7-1 and
IFN-g were able to induce a stronger specific antitumor
immune response in vitro. Mice with unmodified or single
gene–modified A-549 cells developed discernible tumors
at the second week after inoculation. However, those
injected with A-549/B7-1 þ IFN-g developed discernible
tumors at the third week. After 5 weeks, the mice were all
killed. Spleens were taken from SCID mice that had been
inoculated intraperitoneally with human leukocytes.
Immunoflow cytometry results indicated that the number of
human CD3þ lymphocytes in mice spleen cell suspensions
ranged from 10% to 60%. By direct ELISA, the concentration of human IgG in the reconstituted mice sera ranged
from 1.5 mg/ml to 5 mg/ml.
The B7-1- and IFN-g-modified vaccine was able to
enhance the systemic immune response, resulting in
reduced tumor growth, which demonstrated that the coexpression of B7-1 and IFN-g was sufficient to promote an
antitumor immune response.
Abstracts
SMMC-7721 and the normal liver cells Lo2. Either
pCMV-EGFP or pApoAI-EGFP was formulated as pegylated immuno-lipopolyplexes (PILP), a novel and efficient
gene delivery system. Following intravenous administration
of the PILP in H22 tumor-bearing mice, there was significant EGFP expression in liver, tumor, spleen, brain and
lung in the pCMV-EGFP treated mice, whereas in the
pApoAI-EGFP treated mice there was only gene expression
in liver and tumor and the non-liver organ gene expression
was minimum. This study suggests that the use of the PILP
technology and liver-specific promoter enables efficient
and liver-specific expression of an exogenous gene.
Yurong Hu 1, Hui Wang 1, Yingying Shen 1, Lei Wang 1,
Kazuhiro Oka 2, Zhenzhong Zhang 1*, and Yun Zhang 1 *
1
School of Pharmacy, Zhengzhou University, 100 Science Road,
Zhengzhou 450001, China
2
Department of Molecular and Cellular Biology, Baylor College of
Medicine, One Baylor Plaza, Huston, TX 77030, USA
Fax: þ86-371-67781907; E-mail: [email protected] (Z.Z);
[email protected] (Y. Z.)
Our aim is to establish the feasibility and efficacy of combined nonviral hEGFR and hTERT gene therapy for experimental
human
liver
cancer
with
pegylated
immuno-lipopolyplexes (PILP). Two expression plasmids
encoding a short hairpin RNA directed at nucleotides
2627-2655 of the human EGFR mRNA and directed at
nucleotides 1031-1059 of the human TERT mRNA
(anti-hEGFR pDNA and anti-hTERT pDNA) were formulated as PILP with receptor-specific monoclonal antibody
(mAb), the murine 83-14 MAb to human insulin receptor.
Human hepatoblastoma SMMC-7721 cells were transfected
with these PILP. RT-PCR was used to detect the silencing
of hEGFR mRNA and hTERT mRNA on day 4 after transfection. Cell cycle distribution and apoptotic rate were evaluated by flow cytometry (FCM) on 2, 4, 6 days after
transfection. SMMC-7721 cells were implanted subcutaneouly into SCID mice, and gene therapy was started at day
10 after implantation of 2 106 cells. Tumor growth inhibition rate and survival time of mice were calculated.
Immunohistochemistry and Western Blot were used to
detect the hEGFR protein expression in tumor tissue and the
downregulation of hEGFR mRNA was detect by RT-PCR.
A short hairpin RNA inhibited hEGFR gene expression in a
time-dependent manner in cultured hepatoblastoma cells. In
SMMC-7721 cells treated with anti-hEGFR pDNA alone
Po95. Protective effects of ginkgo biloba
extract and dipyridamole injection
against renal ischemia-reperfusion injury
via suppression of jnk pathway in rats
Lanlan Tang 1, Yanhua Zhang 1, Dan Li 2, Shujun Wang 1,
Guanlong Li 1, Yuanyuan Wang 1, Haixia Yao 1,
Shaojie Chen 2, and Wantie Wang 1 *
1
Department of Pathophysiology, Wenzhou Medical College,
Wenzhou, China
2
The People’s Hospital of Yueqing City, Yueqing, China
Ginkgo biloba extract and Dipyridamole injection
(XGD) has protection effect on renal ischemia-reperfusion
injury (IRI) through the JNK pathway, however, the mechanism has not been fully elucidated. We investigated the
expression of JNK in renal IRI in rat kidney tissue, and
study the effect of XGD. Seventy-five male SD rats were
randomly divided into five groups: sham-operated group
(n ¼ 15), IRI group (n ¼ 15), low, middle and high concentration XGD groups (n ¼ 15, respectively). For IRI,
low, middle and high concentration XGD groups, we
removed the right kidney, clamped the left renal pedicle for
1h, then removed the arterial clip and reperfused for 1 h.
Besides, for low, middle and high concentration XGD
groups, before operation XGD was given by tail vein injection for a week, the dosage was 0.9, 1.8, 3.6 mg/kg/d,
respectively. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in renal tissue and serum
creatinine (Scr), blood urea nitrogen (BUN) levels were
detected. Morphologic changes of renal were observed by
Po94. Combined hEGFR and hTERT
gene therapy for experimental human
liver cancer
and co-treated with anti-hEGFR pDNA and anti-TERT
pDNA, it displayed sequence specific silencing of hEGFR
(69.11% and 91.12%, respectively). The percentage of cells
in G0/G1 phase was increased by 17.62% and 45.62%,
respectively, compared with controls; the total apoptotic rate
was 12.47% and 29.59%, respectively. In the two groups,
i.v. anti-hEGFR pDNA and con-i.v. anti-hEGFR pDNA and
anti-TERT pDNA, these plasmids all formulated as PILP
with 8314 MAb. Every five days i.v. RNAi gene therapy
resulted in 66.08% and 90.66% suppression of hEGFR
mRNA, 65.72% and 86.73% of down-regulation of EGR
protein production, 46.13% and 74.04% of tumor growth
inhibition rate, and 94.12% and 135.29% increase in survival time of mice with liver cancer. Combined hEGFR and
hTERT gene therapy could cause stronger suppression for
human hepatoblastoma SMMC-7721 cells growth in vivo
and in vitro compared to hEGFR gene therapy alone.
Abstracts
optical and electronic microscope. Gene expression of JNK
was measured by reverse transcription- polymerase chain
reaction and western bloting was used to detect the protein
expression and activation. Compared with the sham group,
the levels of Scr (214.2 + 40.1), BUN (8.75 +1.28) and
MDA (11.27 + 2.43) of IRI group were significantly
increased (P , 0.01); SOD activity (103.62 + 6.59) was
significantly decreased (P , 0.01); JNK mRNA expression
was sifnificantly higher (P , 0.01); and the phosphorylation of JNK was sifnificantly increased (P , 0.01). After
intervention of XGD, the contents of Scr, BUN and MDA
were significantly lower, SOD activity was significantly
increased; JNK mRNA expression was significantly
reduced, and the phosphorylation of JNK was significantly
inhibited. The different concentration XGD groups showed
no significant difference. Our data suggest that XGD
reduces the expression of JNK mRNA and inhibits the
phosphorylation of JNK to improve renal IRI in rats with
renal function and reduce the extent of injuries.

Poster Presentations Session 1: Environment and Health

Transcription

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