摘要
目的探讨PEG启动子调控腺相关病人毒介导的黑色素瘤分化相关基因MDA-7对肝癌细胞的选择性凋亡诱导效应。方法以重组腺相关病人毒rAAV-PEG-MDA-7表达系统感染人肝癌细胞株HepG2细胞和正常人肝细胞株L02细胞，Westerm Blot检测转染细胞内MDA-7蛋白，MTT法检测细胞增殖抑制率，流式细胞术分析细胞周期、Annexin—V、线粒体跨膜电位（△ψm），RT—PCR检测bcl-2基因mRNA。结果重组腺相关病毒rAAV-PEG-MDA7可特异性转染HepG2细胞，MDA7蛋白在HepG2细胞中高效表达，并呈时间依赖性；重组腺相关病毒rAAV-PEG-MDA-7可抑制HepG2细胞增殖并诱导其凋亡，细胞周期分析处于G0／G1期细胞百分比明显增多，处于G2／M期的细胞减少，并且可见到较明显的凋亡峰的形成，从24h开始Annexin-V阳性细胞比例增多，△ψm降低，抗凋亡基因bcl-2mRNA表达降低。而重组腺相关病毒rAAu-PEG-MDA-7对LO2细胞无类似效应。结论构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统可选择性抑制肝癌细胞增殖和诱导其凋亡，其诱导凋亡机制受到bcl-2家族经线粒体途径的调节。
Objective To investigate the selective apoptosis-induclng effect of adeno-associated virus mediated gene transfer of MDA-7 regulated by PEG promoter on human hepatocellular carcinoma ceils. Methods We transduced human hepatocellular carcinoma cell line HepG2 and normal human hepatocytes LO2 with rAAV-PEG-MDA-7 and assessed the cell growth inhibition, cell cycle and apoptosis. MDA-7 protein was detected by Western blotting, cell growth-inhibiting rate evaluated by MTT and cell cycle, Annexin-V labeling and mitochondrial transmembrane potential were determined by flow cytometry. The expression of bcl-2 mRNA was determined by RT-PCR. Results rAAV-PEG- MDA-7 was effectively transfected into HepG2 cells. MDA-7 protein was highly expressed in HepG2 cells in a time-dependent manner, rAAV-PEG-MDA-7 suppressed HepG2 cell growth. The cells accumulated in the gap 0 and gap 1 （G0/G1） phases, while reduced in the gap 2 and mitosis （G2/M） phases of the cell cycle, and the apoptosis peak significantly occurred, rAAV-PEG-MDA-7 increased the rate of Annexin V labeling positive cells while decreased the mitochondrial transmembrane potential of HepG2 cells within 24 h. The expression of anti-apoptotic gene bcl-2 also decreased. However, there were no such effects on normal human hepatocyte LO2. Conclusion rAAV-PEG-MDA-7 selectively inhibits growth and induce apoptosis of human hepatocellular carcinoma cell line. The apoptosis-inducing mechanism is mediated through intrinsic cell apoptotic pathway involved with the gene regulation of the bcl-2 family.
出处
《中华肝胆外科杂志》
CAS
CSCD
2008年第5期330-333,共4页
Chinese Journal of Hepatobiliary Surgery
基金
广东省科技计划项目（63089）
广州市医药卫生科技项目（2006-YB-020）
