本课题将在前期研究基础上，建立依靠慢病毒载体稳定表达miR-214前体或反义小分子的小鼠ES细胞株。从正负两个效应方面深入探讨miR-214在干细胞向VSMCs定向分化过程中的细胞生物学功能；然后构建含miR-214可能靶基因的3’UTR端质粒，进行萤火素酶双荧光报告基因检测，同时观察miR-214介导的生物学效应是否会由于靶基因的上调/下调而改变，从而探讨miR-214与其潜在靶分子的相互作用关系并初步构建miR-214靶基因交互作用的分子网络。最后，我们还将在小鼠股动脉导丝损伤模型的基础上通过局部输注含有miR-214模拟物或GFP对照的慢病毒载体以实现血管损伤局部miR-214的高表达，从在体水平验证miR-214对其相应靶基因的调控作用、对干/祖细胞定向分化VSMCs的作用及其对血管损伤后新生内膜形成的影响。本项目的实施，将为深入认识干细胞定向分化VSMCs的分子机制提供新的理论依

This research project will establish stable expression of miR-214 precursor or antisense mouse ES cell lines by lentiviral vectors on the basis of preliminary studies. First of all, the biological functions of miR-214 in stem cell orientation differentiation to VSMCs will be deeply explored from both positive and negative aspects. Then we will construct 3'UTR plasmid which contains miR-214 potential target genes to do the dual luciferase reporter gene assay. Meanwhile, the change of biological effects of miR-214 due to up or down regulation of target gene will be observed so as to explore the interaction between miR-214 and its potential target molecules. Thus, molecular networks of miR-214 and its target genes will be constructed. Finally, high expression of miR-214 in vascular injury parts will be achieved by local infusion miR-214 mimic or GFP controlled lentiviral vectors on the basis of mouse femoral artery injury model in order to verify the effects of miR-214 on regulatory its target genes, stem/ progenitor cell orientation differentiation to VSMCs and neointimal formation after vascular injury. Implementation of this project will provide in-depth understanding of the molecular mechanisms of stem cell orientation differentiation to VSMCs.