RNA干扰技术沉默Livin基因联合顺铂对宫颈癌Hela细胞凋亡的影响

目的:采用RNA干扰技术阻断Livin基因的表达,并加入不同浓度的顺铂,研究其抑制Hela细胞Livin基因的效果及增强顺铂诱导宫颈癌细胞凋亡的效应。方法:采用脂质体转染法将pGenesil-1-BIRC71、pGenesil-1-BIRC72、pGenesil-1-HK转染宫颈癌Hela细胞,采用荧光定量RT-PCR、Western-blot检测转染前后宫颈癌Hela细胞Livin基因mRNA和蛋白表达的变化,并加入不同浓度的顺铂(3μg/ml、6μg/ml、9.9μg/ml),采用流式细胞术检测不同时间(24 h、48 h)的细胞凋亡率。结果:转染pGenesil-1-BIRC71、pGenesil-1-BIRC72后Hela细胞中Livin mRNA拷贝数明显减少,蛋白质表达水平显著降低;流式细胞术检测24h、48h凋亡率,干扰组细胞显著高于对照组,并随时间延长凋亡率增加;加入顺铂(3μg/ml、6μg/ml、9.9μg/ml)后流式细胞术检测24 h、48 h凋亡率升高,差异有统计学意义(P<0.05),呈时间及剂量依赖性。结论:以Livin基因为靶向的RNA干扰技术可有效地抑制宫颈癌Hela细胞的Livin基因表达,并可增强顺铂诱导凋亡的效应,且具有时间和剂量依赖性。

Effect of RNA interference combined with cisplatin on silencing expression of Livin gene and the apoptosis of Hela cells

Objective:To block the expression of Livin gene by RNA interference combined with cisplatin,investigate the suppressive effect to Livin gene of Hela cells.Methods:Recombinant plasmids-pGenesil-1-BIRC71,pGenesil-1-BIRC72 and pGenesil-1-HK were transfected into Hela cells,and added cisplatin with different concentrations(3 μg/ml,6 μg/ml,9.9 μg/ml) to observe the inhibition of Livin in Hela cells,the expressions of Livin gene mRNA and protein were detected by fluorescence quantitative real-time PCR and Western blot.The apoptosis rate was detected by flow cytometry after 24 hours and 48 hours.Results:After transfection,mRNA and protein levels of Livin gene were obviously reduced,the apoptosis rate in transfection group was significantly higher than that in control group(P<0.05).The apoptosis rate detected by flow cytometry increased significantly after adding cisplatin(3 μg/ml,6 μg/ml,9.9 μg/ml)(P<0.05).The apoptosis rate increased dependent on the dose and time.Conclusion:RNA interference can inhibit the expression of Livin gene in Hela cells and enhance the apoptosis effect induced by cisplatin,presenting time and dose dependence.