短发夹状RNA介导TNF-α基因沉默及抑制细胞凋亡的作用

Silencing TNF-α gene and inhibiting cell apoptosis by constructing eukaryotic expression vector of small hairpin RNA

目的探讨特异性的短发夹状RNA（shRNA）沉默肿瘤坏死因子-α（TNF-α）基因及抑制细胞凋亡的作用效果。方法构建针对大鼠TNF-α基因编码区的短发夹状RNA真核表达载体质粒pRNAT-U6.1-TNF-αshRNA,采用电穿孔的方法转染RK3E细胞,经G418筛选后,形成稳定的表达TNF-αshRNA的细胞系。实验分为3组,⑴正常对照组：未转染的RK3E细胞;⑵阴性对照组：转染空载体pRNAT-U6.1的RK3E细胞系;⑶实验组：转染TNF-αshRNA的RK3E细胞系。经脂多糖（LPS）孵育12h后,流式细胞仪检测各组细胞凋亡率,RT-PCR检测mRNA的表达。结果成功构建大鼠TNF-α基因的短发夹状RNA真核表达载体质粒pRNAT-U6.1-TNF-αshRNA;经LPS刺激后,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）,TNF-α的mRNA含量显著降低（P〈0.05）。结论针对TNF-α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RK3E细胞凋亡。

Objective To investigate the＇effect of RNA interference and apoptosis by silencing TNF-α gene with small hairpin RNA. Methods The eukaryotic expression vector of small hairpin RNA targeting rat TNF-α gene was constructed and transfected into RK3E cell by electroporation. After G418 selection, the stable cell line expressing TNF-α shRNA was constructed. The experiment was divided into 3 groups ：（1）normal control group, RK3E cell without transfection; （2）negative control group, RK3E cell transfected with blank vector; （3）experimental group, RK3E cell transfected with TNF-α shRNA. After incubation with LPS for 12h, the ratio of apoptosis was determined by flow cytometry, the level of mRNA was detected by RT-PCR. Results Small hairpin RNA targeting rat TNF-α gene had been constructed. Compared with the normal control group and negative control group, the ratio of apoptosis in the experimental group was significantly decreased（ P 〈 0. 01 ）, and the expression of TNF-α mRNA was significantly inhibited （ P 〈 0. 05）. Conclusion Hairpin shRNA targeting TNF-α gene can lead to obvious gene silence in vitnt and inhibit cell apoptosis.