雷帕霉素诱导人肝癌细胞BEL-7402凋亡中Bcl-2作用研究

目的探讨雷帕霉素(rapamycin,RAPA)体外对肝癌BEL-7402细胞生长抑制及诱导细胞凋亡中的作用及Bcl-2变化在凋亡机制中的意义。方法以5、10、20、30、40和50nmol/L不同浓度的RAPA作用于体外培养的BEL-7402细胞,MTT法检测细胞生长抑制率,应用流式细胞仪检测细胞凋亡,Hoechst33258荧光染色法观察细胞凋亡时的形态学变化,Westernblot观察Bcl-2、Bcl-xl和bax等凋亡相关表达变化。结果RAPA可显著抑制BEL-7402细胞的生长,诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系。RAPA作用肝癌细胞BEL-740248h后,在Hoechst33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征,凋亡过程中伴有抗凋亡蛋白Bcl-2、Bcl-xl表达的降低和促凋亡蛋白bax上调。结论RAPA可能通过诱导抗凋亡蛋白Bcl-2、Bcl-xl表达的降低和促凋亡蛋白bax表达上调而诱导凋亡发生,抑制BEL-7402细胞的生长。

Role of Bcl-2 in apoptosis induced by Rapamycin on hepatocelluar carcinoma BEL-7402 Cells

OBJECTIVE:To investigate cell growth inhibition and apoptosis induced by rapamycin on human hepatocelluar carcinoma BEL-7402 cells and the role of Bcl-2 in its action mechanism. METHODS: BEL-7402 cells were given in culture medium with rapamycin in different concentrations, 5, 10, 20, 30, 40 and 50 nmol/L. Cell viability was assessed by MTT assay. Cell apoptosis was observed by Hoechst 33258 staining and flow cytometry (FCM).The Bcl-2 apoptotic related genes were assayed by Western blotting. RESULTS: Rapamycin inhibited the growth of BEL-7402 cells and induced apoptosis significantly in time-and dose-dependent manner. Marked morphological changes of apoptosis were observed by Hoechst 33258 staining after the cells were exposed to rapamycin for 48 h. Western blotting analysis demonstrated that anti-apoptotic protein Bcl-2 and Bcl-xl were downregulatd while pro-apoptotic protein bax was upregulated remarkably in a time dependent manner when apoptosis ocurred. CONCLUSION: Rapamycin has significant antiproliferation effect by induction of apoptosis with downregulation of anti-apoptotic protein Bcl-2 , Bcl-xl and upregulation of pro-apoptotic protein bax.