携带mda-基因的复制缺陷型腺病毒通过促进线粒体释放促凋亡蛋白杀伤肝癌细胞  

mda-7-carrying replication-defective adenovirus selectively kills hepatocellular carcinoma cells by facilitating release of proapoptotic protein from mitochondria

作　　者：王从俊[1] 彭志海[1] 郑建伟[2] 陈堃[2] 胡回忆[2] 吉文伟[2] 于愿[2] 薛新波[2] 

机构地区：[1]上海交通大学附属上海市第一人民医院普通外科,200080 [2]华中科技大学同济医学院附属同济医院胆胰外科中心

出　　处：《中华肿瘤杂志》2008年第9期649-653,共5页Chinese Journal of Oncology

基　　金：湖北省科技攻关重点资助项目（2006AA301B52-4）

摘　　要：目的探讨黑色素瘤分化相关基因7／白介素24（mda-7／IL-24）基因选择性杀伤肝癌细胞的机制。方法应用携带mda-7基因的复制缺陷型腺病毒Ad．mda-7感染正常肝细胞l02和肝癌细胞HepG2。通过逆转录聚合酶链反应（RT-PCR）、酶联免疫吸附实验（ELISA）及Westernblot方法，观察mda-7／IL-24基因的表达；应用Hoechst染色和流式细胞仪观察mda-7／IL-24对肝癌细胞的杀伤作用；采用RT—PCR和Westemblot方法，观察Bcl-2家族和caspase-9基因表达的变化，分离不同感染时点细胞内线粒体和胞浆蛋白，并检测线粒体释放促凋亡蛋白细胞色素C（Cyt-C）和Smac／DIABLO的变化过程。结果Ad．mda-7能介导mda-7／IL-24在两种细胞株中的高效表达。能选择性杀伤肝癌细胞，感染24h后，HepG2细胞凋亡率为24．0％±4．6％，而对正常的肝细胞没有影响。RT—PCR和亚细胞蛋白的分析结果显示，胞浆内Bcl-2和Bcl-xL的表达在HepG2细胞中明显下降，而在1-02细胞中的表达无变化，Bax在肝癌细胞中的表达明显增强，Bak的表达无变化；Ad．mda-7能促进细胞线粒体释放Cyt-C和Smac／DIABLO蛋白，并促进caspase-9的表达。结论Ad．mda-7能选择性杀伤肝癌细胞HepG2，通过促进线粒体促凋亡蛋白的释放而诱导肝癌细胞凋亡。Objective To investigate the mechanism that mda-7/IL-24 selectively kills hepatocellular carcinoma （HCC） HepG2 cells in vitro. Methods HCC cell line HepG2 and normal liver cell line L02 were infected with Ad. mda-7. The expression of mda-7/IL-24 was detected by RT-PCR and ELISA, respectively. The apoptotic effects were confirmed by Hoechst staining and flow cytometry assay, respectively. Furthermore, Bcl-2 family proteins, cytochrome C, Smac/DIABLO and caspase-9 were determined by Western blot. Results The exogenous mda-7/IL-24 gene was expressed in HepG2 and L02 cells infected with Ad. mda-7. Ad. mda-7 induced apoptosis in HepG2 but not in L02 cells in vitro. The induction of tumor cell apoptosis is correlated with the increasing expression of Bax and decreasing expression of Bcl-2 and Bcl-xL genes, then facilitated the releasing of cytochrome C and Smac/DIABLO from mitochondria to cytoplasm and increasing the expression of caspase-9, eventually, resulted in apoptosis. Conclusion Ad. mda-7 selectively induces growth inhibition and apoptosis in hepatocellulae carcinoma HepG2 ceils but not in normal L02 hepatocytes in vitro, and the mechanism might involve the decrease of Bcl-2 and Bcl-xL and increase of Bak expression, facilitating the release of cytochrome C and Smac/DIABLO from mitochondria in HCC cells.

关 键 词：MDA-7/IL-24 肝肿瘤 凋亡 细胞色素C SMAC/DIABLO Bcl-2 

分 类 号：R686[医药卫生—骨科学]