RNA干扰抑制血管平滑肌细胞MT1-MMP基因表达的实验研究

Inhibitory Effects of RNA Interference on the Expression of MT1-MMP Gene of Vascular Smooth Muscle Cells

目的探讨膜型基质金属蛋白酶-1(MT1-MMP)shRNA真核表达载体(pGenesil-1-MT1-MMP)对大鼠血管平滑肌细胞(VSMC)MT1-MMP基因mRNA表达及蛋白表达的影响。方法构建质粒表达载体pGenesil-1-MT1-MMP和pGenesil-1-HK;实验分为3组:空白对照组,阴性对照组(加入pGenesil-1-HK),RNA干扰(RNAi)组(加入pGenesil-1-MT1-MMP)。用脂质体瞬时转染VSMC,48 h后通过荧光显微镜观察转染效果,流式细胞仪检测转染效率。于转染后72 h采用RT-PCR方法检测MT1-MMP mRNA表达水平,用Western blot方法检测MT1-MMP蛋白质表达水平。结果瞬时转染VSMC 48 h后通过流式细胞仪检测转染效率高达85.5%,转染pGenesil-1-MT1-MMP质粒72 h后,RNAi组MT1-MMP mRNA及蛋白质水平明显下调(P<0.05)。结论靶向MT1-MMP的短发夹RNA质粒能够高效地转染入VSMC,并能有效下调MT1-MMP基因mRNA和蛋白的表达。

Objective To explore whether MT1-MMP shRNA eukaryotic plasmid expression vector（pGenesil-1-MT1-MMP）could be effectively transfected into vascular smooth muscle cells（VSMC）and inhibit the MT1-MMP gene expression in VSMC.Methods pGenesil-1-MT1-MMP and pGenesil-1-HK were designed and synthesized.The recombinant plasmids were transiently transfected into VSMC through Lipofectamine 2000.Three groups were assigned：blank control group,negative control group（pGenesil-1-HK）,and RNAi group（pGenesil-1-MT1-MMP）.Forty-eight h after transfection,the transfection effect was confirmed by fluorescence microscope.The transfection efficiency was tested by flow cytometry.Seventy-two h after transfection,the expression levels of MT1-MMP mRNA and protein were detected by semi-quantitative reverse transcription PCR（RT-PCR）and Western blot,respectively.Results The pGenesil-1-MT1-MMP plasmid vector was successfully tranfected into VSMC.The transfection efficiency at 48th h was 85.5%.Seventy-two h after transfection of pGenesil-1-MT1-MMP,the expression levels of MT1-MMP mRNA and protein were down-regulated significantly（P〈0.05）.Conclusion The MT1-MMP specific shRNA expression plasmid constructed in vitro was successfully transfected into VSMC.pGenesil-1-MT1-MMP could effectively down-regulate the expression of MT1-MMP gene.