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目的 探讨稳定过表达Smac基因对胃癌细胞凋亡的影响。方法 脂质体介导Smac基因转染MKN- 45细胞,G418筛选阳性克隆。逆转录聚合酶链反应(RT -PCR)和Western印迹鉴定Smac表达,同时以丝裂霉素作为凋亡诱导因子,采用锥虫蓝活细胞拒染法检测癌细胞体外生长活性,透射电镜、吖啶橙溴化乙啶荧光染色法、末端TdT酶标记技术(TUNEL)观察癌细胞凋亡及比率;Western印迹、比色法检测细胞内caspase- 3表达。结果 所获胃癌亚克隆细胞MKN -45 /Smac的SmacmRNA、蛋白表达水平均显著高于MKN -45(P<0 01)。10μg/ml丝裂霉素作用6 ~24h后,与MKN -45细胞比较,MKN 45 /Smac细胞生长活性减弱10 .0% ~30 8% (P<0 .01),部分MKN- 45 /Smac细胞发生典型的凋亡形态学改变,凋亡率增高21 .2% (P<0. 01 )。丝裂霉素作用后,MKN -45 /Smac细胞内caspase- 3的表达和活性均较MKN- 45细胞显著增强(P<0 .01)。结论 胃癌细胞中Smac基因的过表达,能提高丝裂霉素作用后细胞中caspase 3的表达和活性,显著诱导癌细胞凋亡,为调控胃癌细胞凋亡活性提供了新的途径。
Objective To investigate the effect of Smac gene overexpression on gastric cancer cell apoptosis. METHODS: Smac gene was transfected into MKN-45 cells by Lipofectamine 2000, and positive clones were screened by G418. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of Smac. At the same time, mitomycin was used as an inducer of apoptosis. The growth activity of cancer cells in vitro was detected by trypan blue exclusion method. Transmission electron microscopy, Methyl orange bromide ethidium bromide staining and TUNEL were used to observe the apoptosis rate of cancer cells. The expression of caspase-3 was detected by Western blot and colorimetric assay. Results The SmacmRNA and protein expression levels of MKN-45 / Smac in gastric cancer subclone were significantly higher than those in MKN-45 (P <0.01). Compared with MKN-45 cells, the growth activity of MKN 45 / Smac cells decreased by 10 .0% ~ 30 8% (P <0.01) and some of MKN-45 / Smac A typical morphological changes of apoptosis occurred in the cells, the apoptosis rate increased by 21.2% (P <0.01). After mitomycin treatment, the expression and activity of caspase-3 in MKN-45 / Smac cells were significantly higher than those in MKN-45 cells (P <0.01). Conclusion The overexpression of Smac gene in gastric cancer cells can increase the expression and activity of caspase 3 in cells after mitomycin C treatment, and induce the apoptosis of cancer cells significantly, which provides a new way to regulate the apoptosis of gastric cancer cells.