Smac基因过表达联合顺铂对SMMC-7721细胞增殖和凋亡的影响

目的 探讨Smac基因过表达联合顺铂对肝癌细胞增殖和凋亡的影响.方法 采用脂质体介导的转染方法 ,将重组质粒pcDNA3.1+hSmac导入人肝癌细胞株SMMC-7721中,采用Western blot和流式细胞术检测转染前后Smac蛋白表达情况.转染24 h后分别加入终浓度为5、15、25 μg/ml的顺铂诱导细胞凋亡.采用四甲基偶氮唑盐比色法检测癌细胞的增殖抑制作用,吖啶橙-溴化乙锭荧光染色法和膜联蛋白V/碘化丙啶双染标记流式细胞术检测细胞凋亡. 结果 Westernblot和流式细胞术检测结果证实转染后Smac蛋白表达明显增加(P＜0.01).与空白对照相比,Smac基因过表达可抑制癌细胞增殖,促进凋亡(P＜0.01).而且给予顺铂处理后,与空白对照组相比,细胞生长抑制率随剂量增加而显著上升,且转染Smac的细胞生长抑制率较相应未转染Smac的细胞明显升高(P＜0.01).吖啶橙-溴化乙锭荧光染色法和流式细胞术检测显示,转染Smac加顺铂处理组较单纯顺铂处理组细胞凋亡明显增加,差异具有统计学意义(P＜0.01).这表明Smac基因过表达可增强顺铂对肝癌细胞的增殖抑制和凋亡促进作用. 结论 促凋亡基因Smac可在肝癌细胞中过表达,抑制癌细胞的增殖和促进凋亡;而且过表达的Smac基因可增强癌细胞对化疗药物顺铂的敏感性,这为研究Smac在癌细胞凋亡过程中的调控作用以及肝癌化学治疗效果的改善提供了实验基础.

Effects of over-expressed Smac gene coupling with cisplatin on proliferation and apoptosis of hepatocarcinoma cells

OBJECTIVE: To investigate the effects of over-expressed Smac gene combined with cisplatin (CDDP) on proliferation and apoptosis of hepatic carcinoma cells. METHODS: The recombinant plasmid pcDNA3.1+-hSmac was introduced into the human hepatic carcinoma SMMC-7721 cells using a liposome-mediated method. The expression of Smac protein was detected by Western blot and flow cytometry. The cells were treated with three different doses of CDDP, 5, 15 and 25microg/ml, for 24 hours after the transfection. MTT colorimetry was used to detect the cellular growth-inhibitory effects; acridine orange-ethidium bromide fluorescent staining (AO/EB) and flow cytometry with annexin V-PI double staining METHODS:were used to detect the changes of cell apoptosis. RESULTS: Western blot and flow cytometry results demonstrated that the Smac protein level in SMMC-7721 cells was significantly increased after the transfection (P less than 0.01). Compared with that of the control group, the over-expressed Smac gene inhibited the cell growth and induced cell apoptosis (P less than 0.01). After being treated with CDDP, the inhibitory rates were increased significantly with increasing concentrations of CDDP compared with that of the control group, and the inhibitory rate of the CDDP-treated plus Smac group was significantly higher than that of the CDDP-treated group (P less than 0.01). The results detected by AO/EB and flow cytometry demonstrated that the apoptotic rates of CDDP-treated plus Smac group were higher than those of the CDDP-treated group (P less than 0.01). The results demonstrated that the Smac over-expression enhanced the effects of cell growth inhibition and apoptotic promotion induced by CDDP. CONCLUSION: The pro-apoptotic Smac gene could be over-expressed in hepatocarcinoma SMMC-7721 cells and inhibit cell growth and induce apoptosis. Moreover the over-expressed Smac could enhance the chemotherapeutic sensitivity of SMMC-7721 to cisplatin. This experimental work may help in further study on the regulatory mechanism of Smac in apoptosis and improve the chemotherapeutic effect on hepatoma.