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目的:研究Bak基因过度表达在HCC-9204细胞凋亡途径中的作用以及对多柔比星和长春瑞滨的可能的增敏作用.方法:采用MT-Ⅱ可调控性表达载体系统,通过外加ZnSO_4(100 μmol/L)诱导Bak基因表达,并获得稳定转染子.以形态学标准并结合TUNEL或流式细胞仪检测细胞凋亡.克隆形成实验反映克隆细胞存活率,MTF法检测细胞活力.结果:Bak基因过度表达的细胞出现显著的细胞死亡,TUNEL证实为一种凋亡性细胞死亡.流式细胞仪的结果显示Bak能够显著地诱导细胞在G_1期聚积并在阿霉素诱导后24 h 19.26％的细胞发生凋亡.Bak基因过度表达只能显著降低阿霉素处理组的克隆存活率,而对长春花碱组没有显著效果.MTT实验的结果类似,提示Bak基因能够选择性对阿霉素诱导的细胞死亡具有增敏作用,而对长春花碱没有这种作用.结论:Bak基因的过度表达使HCC-9204细胞的细胞周期在G_1延长,导致细胞凋亡并选择性对化疗药物具有增敏作用.
OBJECTIVE: To investigate the role of Bak gene overexpression in the apoptotic pathway of HCC-9204 cells and its possible sensitizing effects on doxorubicin and vinorelbine.Methods: MT-Ⅱ was used to regulate expression vector system, The expression of Bak gene was induced by ZnSO_4 (100 μmol / L), and the stable transfectants were obtained.The cell apoptosis was detected by morphological criteria combined with TUNEL or flow cytometry.The clone formation assay reflected the survival rate of cloned cells and the cell viability Results: Significant cell death was observed in cells overexpressing Bak gene and TUNEL proved to be a kind of apoptotic cell death.The results of flow cytometry showed that Bak can significantly induce cell accumulation in G 1 phase and induce apoptosis after adriamycin induction 24 h 19.26% of the cells were apoptosis .Bak gene overexpression can only significantly reduce the cloned survival rate of doxorubicin treatment group, but no significant effect on the vinblastine group.MTT test results are similar, suggesting Bak gene can be selectively Which could sensitize doxorubicin-induced cell death but not vinblastine.Conclusion: Overexpression of Bak gene prolongs cell cycle in HCC-9204 cells and leads to apoptosis Sensitization of having a selective chemotherapeutic agents.