CAJ | 학술논문

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Western blot方法,观察mda-7/IL-24基因的表达;应用Hoeehst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Western blot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24 h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Bel-2和Bcl-xL的表达在HepG2细胞中明显下降,而在L02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.
목적 탐토흑색소류분화상관기인7/백개소24(mda-7/IL-24)기인선택성살상간암세포적궤제.방법 응용휴대mda-7기인적복제결함형선병독Ad.mda-7감염정상간세포L02화간암세포HepG2.통과역전록취합매련반응(RT-PCR)、매련면역흡부실험(ELISA)급Western blot방법,관찰mda-7/IL-24기인적표체;응용Hoeehst염색화류식세포의관찰mda-7/IL-24대간암세포적살상작용;채용RT-PCR화Western blot방법,관찰Bcl-2가족화caspase-9기인표체적변화,분리불동감염시점세포내선립체화포장단백,병검측선립체석방촉조망단백세포색소C(Cyt-C)화Smac/DIABLO적변화과정.결과 Ad.mda-7능개도mda-7/IL-24재량충세포주중적고효표체.능선택성살상간암세포,감염24 h후,HepG2세포조망솔위24.0%±4.6%,이대정상적간세포몰유영향.RT-PCR화아세포단백적분석결과현시,포장내Bel-2화Bcl-xL적표체재HepG2세포중명현하강,이재L02세포중적표체무변화,Bax재간암세포중적표체명현증강,Bak적표체무변화;Ad.mda-7능촉진세포선립체석방cyt-C화Smac/DIABLO단백,병촉진caspase-9적표체.결론 Ad.mda-7능선택성살상간암세포HepG2,통과촉진선립체촉조망단백적석방이유도간암세포조망.
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. Theinduction of tumor ceU 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 cells 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.