【摘要】 目的筛选针对STAT5A基因有效的siRNA,研究抑制STAT5A基因的表达对人肝癌HepG2细胞增殖、周期及凋亡的影响,探讨STAT5A基因在肝癌发生发展中的作用。方法设计并化学合成针对STAT5A基因三个靶点的siRNA,采用脂质体法转染人肝癌细胞HepG2,分别用半定量RT-PCR、Western blot技术检测转染后HepG2细胞STAT5A mRNA和蛋白表达的变化,从中筛选出一段干扰效率最显著的siRNA;用该siRNA转染HepG2细胞,MTT法检测细胞增殖情况,流式细胞仪检测细胞凋亡率。结果三段STAT5A基因的siRNA均对HepG2细胞中STAT5A mRNA和蛋白表达产生了不同程度的抑制作用,其中以siRNA-3697的干扰效率最高,STAT5A mRNA和蛋白的表达抑制率分别为72.03%和66.27%(P<0.05);转染后1～4 d细胞生长速度减慢、增殖显著抑制(P<0.05);转染后48 h细胞凋亡率为37.33%(P<0.05)。结论成功筛选出针对STAT5A基因有效的siRNA;该siRNA可特异地阻断HepG2细胞STAT5A基因的表达,抑制细胞生长,诱导细胞凋亡。更多还原

【Abstract】 Objective To screen for effective siRNAs for STAT5A gene and study the effect of suppression of STAT5A gene expression on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2, and to explore the role of STAT5A in generation and development of hepatocellular carcinoma.Methods Three siRNAs targeted at STAT5A gene were designed and synthesized chemically, and then transfected into HepG2 cells with liposome transfection method. The expression levels of STAT5A mRNA and protein were detected by semi-quantitative reverse transcript polymerase chain reaction(RT-PCR) and immunoblotting assay(Western blot) respectively, and then the effective sequence was selected based on the interference efficiency. MTT method was used to detect the cell proliferation, and FCM was used to detect the cell apoptosis after the optimal siRNA was transfected. Results The three siRNAs could inhibit STAT5A gene expression at both mRNA and protein levels in various degrees, and siRNA-3697 was the most efficient,with the inhibition rates of STAT5A mRNA and protein expressions of 72.03% and 66.27%(P<0.05),respectively. The result of MTT analysis showed that the cell growth rat of the experiment group was decreased and the proliferation inhibition rate was inhibited significantly after 1-4d of transfection(P<0.05).And FCM assay indicated that the apoptotic rate of the experiment group was 37.33% after 48h of transfection(P<0.05).Conclusion An effectual siRNAfor STAT5Agene was successfully screened out, and this siRNA could inhibit the expression of STAT5A gene efficiently,causing inhibition of the proliferation of HepG2cells, and promotion of apoptosis obviously.更多还原