shRNA对大鼠心肌细胞牛磺酸转运体的抑制作用

Inhibitory Effects of shRNA on Taurine Transporter of Rats Cardiac Myoblasts

本试验旨在研究短发夹RNA(shRNA)对大鼠心肌细胞牛磺酸转运体(TauT)的抑制作用。根据已克隆的大鼠心肌TauT基因序列设计并构建了3个shRNAs(shRNA1#、shRNA2#、shRNA3#)及1个阴性对照shRNA表达载体,利用脂质体LipofectamineTM2000将构建好的重组质粒shRNA1#、shRNA2#、shRNA3#及阴性对照质粒分别转染H9c2细胞,细胞分为对照组(未转染组)、shRNA-Neg组(阴性对照组)和3个转染组(shRNA1#组、shRNA2#组、shRNA3#组),每组3个重复。应用四唑盐(MTT)法检测H9c2细胞增殖情况,实时荧光定量PCR检测TauT mRNA表达水平。结果表明,与阴性对照组和未转染组比较,shRNA1#质粒仅在转染细胞后的24 h极显著降低了TauT mRNA表达水平(P<0.01),shRNA2#质粒在转染细胞后的24、48、72 h均极显著降低了TauT mRNA表达水平(P<0.01),shRNA3#质粒则是在转染细胞后的72 h极显著降低了TauT mRNA表达水平(P<0.01);MTT检测结果表明,转染TauT shRNA1#、shRNA2#、shRNA3#入H9c2细胞24、48、72 h后,细胞增殖均未受到显著影响(P>0.05)。上述结果提示,shRNA2#重组质粒能有效抑制TauT基因的表达,且短期内未影响H9c2细胞增殖,为研究TauT基因及揭示牛磺酸在心肌细胞代谢中的作用奠定基础。

This experiment was conducted to investigate the inhibitory effects of short hairpin RNA （shRNA）on taurine transporter （TauT） of rats cardiac myoblasts.Three shRNAs expression vectors （shRNA1 #,shRNA2# and shRNA3#） and one negative control shRNA expression vector were constructed to be targeted directly at TauT gene.Then the recombinant plasmids shRNA1#,shRNA2#,shRNA3# and shRNA-Neg were transfected into H9c2 cells with liposomes LipofectamineTM 2000,the cells were divided into control group （untransfected group）,shRNA-Neg group （negative control group）,and three transfected groups （shRNA1# group,shRNA2# group and shRNA3# group）,each group contained three replicates.After transfection,TauT mRNA expression level was examined by using real-time quantitative PCR,and the state of cell proliferation was detected by methyl thiazolyl tetrazolium （MTT） assay.The results showed that compared with the negative control group and untransfected group,the shRNA1# plasmid significantly decreased the TauT mRNA expression level at 24 h after transfection （P 〈 0.01）,the shRNA2# plasmid significantly decreased the TauT mRNA expression level at 24,48 and 72 h after transfection （P 〈0.01）,and the shRNA3# plasmid significantly decreased the TauT mRNA expression level at 72 h after transfection （P 〈 0.01）.The MTT detected result showed that the cell proliferation in the transfected cells was not significantly affected by shRNAs plasmid compared with untransfected cells and negative control cells at 24,48,72 h （P 〉 0.05）.The present study indicates that the shRNA2# recombinant plasmid can effectively inhibit the expression of TauT gene and does not affect the proliferation of H9c2 cells at 72 h after transfection,which will be of benefit to the further study on the functions of TauT and taurine in cardiocytes metabolism.