大鼠心肌素慢病毒RNA干扰载体的构建及其对血管平滑肌细胞分化的影响

Construction of a lentiviral RNA interference vector targeting rat myocardin and its effect on vascular smooth muscle cell differentiation

目的:构建大鼠心肌素(myocardin)慢病毒RNA干扰(RNAi)重组质粒,分析该RNAi质粒的干扰效果及其对血管平滑肌细胞(VSMCs)分化的影响。方法:设计并合成3对针对大鼠myocardin的shRNA片段,退火后将双链oligo-DNA克隆入慢病毒质粒载体pGCSIL-GFP获得重组干扰质粒pGCSIL-GFP-shMyocd。以pEGFP-N1/X124G表达载体构建含Flag标签的myocardin表达质粒pEGFP-N1-Myocd。将重组干扰质粒和过表达质粒共转染293T细胞,Western blotting分析Flag标签蛋白的表达,筛选出干扰效率最好的干扰质粒并大量扩增。干扰质粒转染大鼠主动脉VSMCs,RT-PCR和Western blotting检测myocardin及分化标志物SM22α的表达。结果:成功构建大鼠myocardin的慢病毒干扰质粒pGCSIL-GFP-shMyocd和过表达质粒pEGFP-N1-Myocd。上述2种质粒共转染293T细胞后,1号干扰质粒转染组Flag标签蛋白表达下降最显著。将1号质粒进行大包装,获得滴度为1×10^(12)TU/L的慢病毒颗粒,该病毒转染VSMCs后,myocardin表达明显下调并伴随分化标志物SM22α表达的显著下降。结论:成功构建大鼠myocardin慢病毒重组干扰质粒pGCSIL-GFP-shMyocd;抑制基因myocardin表达后伴随VSMCs分化的表达下降,提示myocardin在VSMCs分化过程中的重要性。pGCSIL-GFP-shMyocd重组质粒的成功构建为后续研究特定病理条件下(如动脉粥样硬化)VSMCs表型转变的分子机制奠定了基础。

AIM：To construct a lentiviral RNA interference（RNAi）vector targeting rat myocardin mRNA and to investigate its effect on the differentiation of vascular smooth muscle cells（VSMCs）.METHODS：Three pairs of dsDNA targeting rat myocardin mRNA were designed,synthesized and cloned into lentiviral vector pGCSIL-GFP to generate pGCSIL -GFP-shMyocd lentvirus.A Flag-tagged myocardin-overexpression vector pEGFP-N1-Myocd was constructed with pEGFP-N1/X124G.After these two vectors were cotransfected into 293T cells,the flag protein was assessed by Western blotting to analyze the knockdown effect of pGCSIL-GFP-shMyocd.The expression of myocardin and SM22αwas also detected by RT-PCR and Western blotting after the pGCSIL-GFP-shMyocd viruses were transfected into primary cultured rat aortal VSMCs.RESULTS：The rat myocardin lentviral RNAi vector pGCSIL-GFP-shMyocd and myocardin -overexpression vector pEGFP-N1-Myocd were successfully constructed.After these two kinds of vectors were cotransfected into 293T cells,the No.1 interfering vector displayed the highest inhibitory effect on flag expression.After the No.1 lentvirus at the titer of 1×10^（12）TU/L was transfected into VSMCs,the myocardin and SM22αexpression was significantly attenuated.CONCLUSION：The lentiviral pGCSIL-GFP-shMyocd RNAi vector is successfully constructed, which is useful for further study regarding the molecular mechanism of the phenotypic switching in VSMCs under special pathological conditions such as atherosclerosis.Inhibition of myocardin expression in VSMCs leads to the decrease in the expression of differentiation marker,and implies a crucial role of myocardin in VSMCs differentiation.