摘要
目的:研究一株实验室减毒水泡性口炎病毒(VSV)对HepG2细胞的凋亡诱导作用,并探讨其可能的作用机制.方法:首先将水泡性口炎病毒以1.0感染复数(MOI)的接种密度感染HepG2细胞,同时设接种相同体积培养液的Mock感染组,在不同时间点收集细胞,MTT法检测细胞增殖活性;AO/EB结合Hoechst/PI染色观察细胞凋亡形态学变化;AnnexinV-FITC/PI双染法检测早期凋亡细胞数目;PI染色结合细胞周期分析sub-G1凋亡峰值;JC-1染色测定细胞线粒体跨膜电位(ΔΨm)水平;比色法检测caspase-3,caspase-8及caspase-9的活性变化.结果:减毒VSV感染HepG2细胞后,随着感染时间的增加,HepG2细胞存活率明显降低.VSV感染HepG2细胞24h后,早期凋亡细胞数高于Mock感染组(26.46%±6.01%vs4.86%±2.28%,t=-5.817,P<0.01);sub-G1峰细胞数高于Mock感染组(14.07%±3.83%vs3.99%±1.36%,t=-4.293,P<0.05);HepG2细胞线粒体跨膜电位(ΔΨm)明显降低(t=-4.586,P<0.05);caspase-9和caspase-3的活性显著升高(P<0.05或P<0.01).结论:人肝癌细胞HepG2是VSV敏感细胞株,VSV感染HepG2导致细胞线粒体△Ψm下降,激活caspase-9进而活化下游caspase-3,最终通过内源性线粒体通路诱导细胞凋亡.
AIM：To investigate the apoptosis-inducing effect of a laboratory-attenuated vesicular stomatitis virus（VSV） strain on HepG2 cells and to explore the underlying mechanisms.METHODS：After HepG2 cells were infected with VSV at a multiplicity of infection（MOI） of 1.0,cell viability was determined by MTT assay morphological assessment of apoptosis was performed by acridine orange（AO）/ethidium bromide（EB） and Hoechst/PI staining;apoptotic cells were quantified by annexin V/PI doublestaining and cell cycle analysis;mitochondrial membrane potential（ΔΨm） was measured by JC-1 staining;and activation of caspase proteolytic cascade was measured with caspase-9,caspase-8 and-3 colorimetric assay kits.RESULTS：The attenuated VSV strain could markedly inhibit HepG2 cell proliferation in a time-dependent manner.After HepG2 cells were exposed to VSV at an MOI of 1.0 for 24 h,the percentages of early apoptotic cells（26.46% ± 6.01% vs 4.86% ± 2.28%,t =-5.817,P 0.01） and cells in sub-G1 phase（14.07% ± 3.83% vs 3.99% ± 1.36%,t =-4.293,P 0.05） were increased compared with mock-infected cells.VSV infection significantly decreased mitochondria membrane potential（△Ψm）（t =-4.586,P 0.05） and increased the activity of caspase-9 and caspase-3（both P 0.05）.CONCLUSION：Human hepatoma cell line HepG2 is highly susceptible to infection with oncolytic VSV.VSV can inhibit the proliferation of HepG2 cell and promote apoptosis through the intrinsic mitochondria pathway.VSV-induced collapse of the mitochondrial trans-membrane potential could exert a feedback effect to elicit caspase-9,and then lead to the activation of the key downstream factor caspase-3.
出处
《世界华人消化杂志》
CAS
北大核心
2011年第7期667-673,共7页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No. 31001070
吉林省科技发展计划基金资助项目
No. 20100146~~
关键词
水泡性口炎病毒
肝细胞癌
凋亡
HEPG2细胞
Vesicular stomatitis virus
Hepatocellular carcinoma
Apoptosis
HepG2 cells
