RNA干扰对大鼠血管内皮细胞血管紧张素转换酶表达的抑制作用

目的用RNA干扰技术选择性下调大鼠血管内皮细胞(ECs)上血管紧张素转换酶(ACE)的表达。方法构建携带ACE基因特异短发夹RNA(shRNA)编码序列的质粒载体pACE-shRNA,与脂质体转染剂制备成不同比例的复合物,转染原代培养的大鼠主动脉ECs,在荧光显微镜下检测转染效率,用四甲基氮唑盐比色法检测细胞存活率,选择出质粒与转染剂的最佳配比。然后以最佳配比复合物转染ECs,分别于转染前及转染后24、48、72h通过半定量RT-PCR和Western blot法检测ACE的表达。结果质粒与转染剂的最佳配比为质粒1.0μg、转染剂4μl,此时转染效率为(81.2±6.2)%,细胞生存率为(90.2±4.1)%。以最佳配比复合物转染ECs后24h,ACE的mRNA和蛋白质表达无明显变化;转染后48h,表达明显降低,差异有统计学意义(P<0.01);72h时表达更低,只有微弱的条带显示。结论RNA干扰能选择性下调原代培养的大鼠血管ECs上ACE的表达,这可能为心血管疾病基因治疗的研究和应用提供新方法。

Inhibition of angiotensin-converting enzyme expression in rat vascular endothelial cells by RNA interference

Objective To selectively knockdown the expression of angiotensin-converting enzyme(ACE) in rat vascular endothelial cells(ECs) by RNA interference.Methods Recombinant plasmids expressing ACE gene-specific shRNA(pACE-shRNA) were constructed.Primary cultures of rat aortic endothelial cells were transfected with pACE-shRNA with different ratios of transfection reagent.Transfection efficiency was determined by fluorescence microscopy and cell viability was calculated by MTT assay.Rat aortic endothelial cells were then transfected with the optimal condition.Total RNA and total protein were extracted from endothelial cells before transfection and 24,48,72 hours after transfection.ACE mRNA and protein expression were analyzed by semi-quantitative RT-PCR and Western blot.Results The optimal ratio of pACE-shRNA to transfection reagent was 1.0 μg to 4 μl.With this transfection condition,transfection efficiency was(81.2±6.2)% and cell viability was(90.2±4.1)%.Expression of ACE mRNA and protein in endothelial cells were significantly reduced 48 hours after pACE-shRNA transfection,and was nearly undetectable after 72 hours.Conclusions RNA interference can knockdown ACE expression in cultured Ecs,which maybe provide a new gene therapy tool for cardiovascular diseases.