转染Smac对肝癌细胞凋亡的影响

Effect of ectopic Smac/DIABLO overexpression on cell apoptosis and growth of hepatocellular carcinoma HepG2 cells

目的探讨过表达Smac基因对人肝癌HepG2细胞凋亡、细胞周期及凋亡相关蛋白Survivin、半胖氨酸蛋白酶-3(Caspase-3)表达的影响。方法从人K562细胞中扩增Smac cDNA,构建含Smac cDNA的真核表达载体pEGFP-C1-Smac,并转染人肝癌细胞HepG2。流式细胞仪检测分析细胞凋亡百分率,凋亡相关蛋白Survivin、Caspase-3的表达及细胞周期。结果转染Smac基因组、转染空载体组、未转染组细胞凋亡率差异有统计学意义(P<0.05)。转染Smac基因组细胞凋亡率高于未转染组,也高于转染空载体组细胞,差异均有统计学意义(P<0.05)。转染Smac基因组细胞Survivin的表达显著高于未转染组,也显著高于转染空载体组细胞,差异均有统计学意义(P<0.05)。未转染组、转染空载体组、转染Smac基因组中Caspase-3蛋白表达差异无统计学意义(P>0.05)。未转染组、转染空载体组、转染Smac基因组HepG2细胞的G0/G1期、S期、G2/M各期细胞比例差异无统计学意义(P>0.05)。结论过表达Smac可诱导人肝癌HepG2细胞凋亡率增加,促进细胞凋亡。

Objective To observe the effect of ectopic overexpression of second mitochondria-derived activator of Caspases（Smac/DIABLO） on cell apoptosis,cell cycle and to investigate the expression of Survivin and Caspase-3 proteins in hepatocellular carcinoma HepG2 cells.Methods The cDNA fragment of human full-length Smac gene was amplified by reverse transcription-PCR from total RNA of the human myeloid leukaemic K562 cell line.The PCR product was ligated with a plasmid vector named pEGFP-C1.The pEGFP-C1 Smac plasmid was confirmed by using restriction enzyme digestion,PCR and DNA sequencing.The recombinant plasmid was transfected into HepG2 cell line by lipofectamine reagent.The expression of full-length Smac was determined by fluorescence microscopy.The apoptosis rates,the expressions of Survivin,Caspase-3 protein and cell cycling were detected by flow cytometry.Results Smac transfectants for 24 hours,the apoptosis rate was significantly higher than that of the untransfected cells,and that in free vector transfected group（P〈0.05）.The expression of Survivin in transfected cells was significantly higher than that（181.96±26.37） of the untransfected groups,and that in free vector transfected group（P〈0.05）.But there was no significant difference in the expression of Caspase-3 among the groups（P〉0.05）.The differences of the population of G0/G1 phase,S phase and G2/M phase in the three groups were not significant（P〉0.05）.Conclusion Cytosolic overexpression of Smac in HepG2 cells can increase apoptosis rate and promote apoptosis.