RNAi介导Caspase-3基因及其抑制LPS诱导肾脏上皮细胞凋亡的研究

Silence of Caspase-3 gene by construction eukaryotic expression vector of small hairpin RNA can inhibit cell apoptosis induced by LPS in kidney epithelial cells

目的:探讨RNA干扰Caspase-3基因及抑制LPS诱导的肾脏上皮细胞凋亡的作用效果。方法:构建针对大鼠Caspase-3基因编码区的短发夹状RNA(shRNA)真核表达载体质粒pRNAT-U6.1-Caspase-3shRNA,用电穿孔法转染RK3E细胞,经G418筛选后,形成稳定的表达Caspase-3 shRNA的细胞系。实验分为3组:①正常对照组:未转染的RK3E细胞;②阴性对照组:转染空载体pRNAT-U6.1的RK3E细胞系;③实验组:转染Caspase-3 shRNA的RK3E细胞系。经脂多糖(LPS)孵育12小时后,流式细胞仪检测各组细胞凋亡率,RT-PCR检测mRNA的表达,以及各组Caspase-8蛋白的活性。结果:与正常对照组和阴性对照组相比,实验组的细胞凋亡率显著降低(P<0.01),Caspase-3的mRNA水平和蛋白活性显著降低(P<0.01)。结论:针对Caspase-3的特异性短发夹RNA可以明显引起靶基因的沉默,进而抑制LPS诱导的肾脏上皮细胞凋亡的发生。

Objective：To investigate RNA interference and apoptosis induced by LPS in kidney epithelial cell through silencing Caspase-3 gene with small hairpin RNA. Methods：We constructed the eukaryotic expression vector of small hairpin RNA targeting rat Caspase-3 gene, and transfected RK3E cell by electroporation. After G418 selection ,the stable cell line expressing Caspase-3 shRNA was obtained, the experiment was divided into 3 groups of： （1）normal control group, RK3E cells without transfection; （2）negative control group, RK3E cells transfected with blank vector; （3） experimental group, RK3E cells transfected with Caspase-3 siRNA. After incubation with LPS for 12 h, the ratio of apoptosis was tested by flow cytometry, the level of mRNA was tested by RT-PCR and Real time PCR, and the Caspase-3 activity was test by kit. Results：Compared with the normal control and negative control group,the ratio of apoptosis was sig- nificantly decreased in experimental group（ P 〈 0. 01 ）, and its activity was significantly decreased also. Conclusion：Hairpin shRNA targeting Caspase-3 gene can lead to obvious gene silence in vitro and inhibit the cell apoptosis induced by LPS in kidney epithelial cells.