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.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
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）
