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

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

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。

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 transfeetion 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.