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目的研究第二线粒体源的半胱天冬酶激活物(Smac)基因联合顺铂对人肝癌细胞SMMC-7721中半胱氨酸天冬氨酸蛋白酶(caspase)-3、caspase-9和细胞色素c(Cyt c)蛋白表达的影响。方法利用脂质体介导的方法将Smac转染入SMMC-7721,G418筛选后采用逆转录-聚合酶链式反应(RT-PCR)和蛋白印迹(western blot)法检测筛选所得SMMC-7721/Smac细胞中Smac mRNA和蛋白的表达,四甲基偶氮噻唑蓝(MTT)法绘制细胞生长曲线;顺铂处理后采用免疫细胞化学法检测细胞中caspase-3、caspase-9和Cyt c蛋白的表达。结果 SMMC-7721/Smac细胞中Smac mRNA和蛋白表达均明显增加,12～120 h A490值为0.28～0.70,均明显低于同一时间点的SMMC-7721(0.31～1.10)(P<0.05);与SMMC-7721比较,SMMC-7721/Smac细胞中caspase-3和Cyt c蛋白表达均增高,累积光密度值(IOD)分别从19 939/14 924增至28 347/21 017(P<0.05);顺铂作用下,SMMC-7721和SMMC-7721/Smac细胞中caspase-3、caspase-9和Cyt c蛋白表达均增加,且后者更明显;剂量为25μg/mL时,SMMC-7721/Smac的caspase-3、caspase-9的IOD分别从对照的28 347/22 412增至46 696/39 728(P<0.001)。结论 Smac稳定过表达联合顺铂可明显增强细胞中凋亡相关蛋白caspase-3、caspase-9和Cyt c表达,从而促进细胞凋亡。
Objective To investigate the effect of the second mitochondrial derived Smac gene and cisplatin on the expression of caspase-3, caspase-9 and cytochromes in human hepatoma cell line SMMC-7721 c (Cyt c) protein expression. Methods SMMC-7721 was transfected into SMMC-7721 cells by liposome-mediated method. After G418 selection, RT-PCR and Western blotting were used to screen SMMC-7721 / The expression of Smac mRNA and protein in Smac cells and the cell growth curve were obtained by MTT assay. The expressions of caspase-3, caspase-9 and Cyt c in cells were detected by immunocytochemistry after cisplatin treatment expression. Results The expression of Smac mRNA and protein were significantly increased in SMMC-7721 / Smac cells. The values of A490 at 12-120 h were 0.28-0.70, which were significantly lower than those at the same time point (0.31-1.10) (P <0.05). Compared with SMMC-7721, the expression of caspase-3 and Cyt c in SMMC-7721 / Smac cells were increased, and the cumulative optical density (IOD) increased from 19 939/14 924 to 28 347/21 017 (P 0.05) ; The expression of caspase-3, caspase-9 and Cyt c in SMMC-7721 and SMMC-7721 / Smac cells increased after cisplatin treatment, and the latter was more obvious; SMMC-7721 / Smac The IOD of caspase-3 and caspase-9 increased from 28 347/22 412 in the control to 46 696/39 728 (P <0.001), respectively. Conclusion Stable overexpression of Smac combined with cisplatin can significantly enhance the expression of caspase-3, caspase-9 and Cyt c in cells and promote the apoptosis.