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目的:观察livin基因的siRNA对人胃癌细胞株BGC823生长与功能的影响。方法:构建针对livin基因siR-NA真核表达质粒pGPU6/GFP/Neo-livin,通过脂质体介导转染至BGC823细胞,半定量RT-PCR、蛋白质印迹法分别检测livin基因mRNA和蛋白表达,激酶活性检测试剂盒检测胱冬肽酶-3(Caspase-3)的活性,MTT法分别检测PBMC和BGC823细胞增殖活性,流式细胞仪检测细胞凋亡率,ELISA法检测混合细胞培养上清中TNF-α分泌量的变化。结果:转染48 h实验组细胞livin mRNA和蛋白表达水平明显受到抑制,表达抑制率分别为64.63%和62.80%,P<0.05;Caspase-3活性增强,细胞凋亡率为22.4%,P<0.05;PBMC和BGC823细胞增殖减慢;细胞培养上清中TNF-α分泌量明显降低,P<0.05。结论:通过抑制BGC823细胞中livin的表达,Caspase-3的活性增加,从而诱导细胞凋亡率上升;livin基因表达沉默可以抑制PBMC细胞增殖,TNF-α分泌减少,提示了livin基因可能是在细胞发生癌变初期过度表达的一个凋亡抑制基因。
Objective: To observe the effect of siRNA of livin gene on the growth and function of human gastric cancer cell line BGC823. Methods: The eukaryotic expression plasmid pGPU6/GFP/Neo-livin for siR-NA of livin gene was constructed and transfected into BGC823 cells by liposome. Semi-quantitative RT-PCR and Western blot were used to detect the expression of livin mRNA and protein, respectively. The kinase activity assay kit was used to detect the activity of caspase-3 (Caspase-3). The proliferative activity of PBMC and BGC823 cells was detected by MTT assay. The apoptosis rate was detected by flow cytometry. The mixed cell culture supernatant was detected by ELISA. Changes in the amount of TNF-α secretion. RESULTS: The expression of livin mRNA and protein was significantly inhibited in the transfected cells for 48 hours. The expression inhibition rates were 64.63% and 62.80%, respectively, P<0.05; Caspase-3 activity was enhanced, and the apoptosis rate was 22.4%. P< 0.05; PBMC and BGC823 cell proliferation slowed down; TNF-α secretion in cell culture supernatant was significantly reduced, P<0.05. CONCLUSION: By inhibiting the expression of livin in BGC823 cells, the activity of Caspase-3 is increased and the apoptosis rate is increased. The silencing of livin gene can inhibit the proliferation of PBMC and the secretion of TNF-α, suggesting that the livin gene may be in the cell. An apoptosis-inhibiting gene that is overexpressed in the early stages of canceration.