负调控miR-195促进MSCs细胞对缺血心肌细胞的保护作用及其机制探讨

Mesenchymal stem cells (MSCs) from bone marrow have a potential of multi-lineage differentiation and special biological characteristics. Transplantation of MSCs can improve cardiac function, reduce infarction size and promote cardiomyocyte regeneration. Differentiation into myocardial cells and angiogenesis are considered to be the basis of the therapeutic effect of MSCs. However, the function of MSCs is seriously weakened by increasing apoptosis and decreasing survival rate of MSCs in hypoxic environment post-transplantation. miRNA (microRNA) is a small non-coding RNA molecule (ca. 22 nucleotides) found in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression by base-pairing to a complementary sequence. miR-195 is one of the important miRNAs, which may regulate many role apoptosis proteins including Bcl2，Bcl-w. Our previous studies found that the down-regulating of miR-195 expression may increase apoptosis and decrease survival rate of MSCs in hypoxic environment post-transplantation. This project intends to research function of the down-regulation miR-195 in vivo and in vitro, can which inhibit apoptosis of MSC cells, promote the protection function of MSCs in ischemic myocardium and promote angiogenesis in ischemic myocardium in rats. To explore ways to strengthen the protection of the ischemic heart, and to Lay a foundation for the treatment of myocardial infarction with MSCs transplantation.

有多系分化的潜能以及特异的生物学特性的骨髓间充质干细胞(MSCs)移植能改善心脏功能，减少梗死面积，并有助于心肌细胞再生。向心肌细胞方向分化和形成新生血管被认为是MSCs细胞治疗作用的基础。然而，MSCs移植后在缺血缺氧环境中凋亡增加，存活率降低，严重削弱了MSCs的作用。微小RNA（microRNAs）是一类由20-25个核苷酸组成的非编码RNAs，通过microRNA的子序列以碱基配对的方式结合互补序列来调节基因表达。miR-195是miRNA中重要的一员，目前研究发现其可能的调控的蛋白包括凋亡基因Bcl2，Bcl-w。前期的研究发现负调控miR-195能减少移植后MSC细胞凋亡，增加存活率。本课题拟研究负调控miR-195在体内体外抑制MSC细胞凋亡、促进MSC细胞保护缺血心肌，促进缺血心肌中新生血管的形成中的作用。探寻加强缺血心脏保护的方法，为MSCs细胞移植治疗心肌梗死奠定基础。

本研究结合分子生物学实验、细胞体外实验、在体动物实验等多个层次的方法，观察微小RNA（miR-195）、锌指转录因子GATA-4基因过表达的骨髓间充质干细胞（MSCs）对心肌梗死后缺血心肌细胞的影响。阐明GATA-4与miR-195、B-细胞淋巴瘤基因w（Bcl-w）因子之间的关系，探讨负调控miR-195、过表达GATA-4加强MSCs细胞对梗死后缺血心肌细胞保护作用，我们研究发现： 1. 体外实验结果显示： GATA-4加强MSCs细胞对心肌缺氧细胞的保护作用, 使缺氧24h后的心肌细胞凋亡和缺氧36h后的心肌细胞损伤进一步减少。2. 体内实验结果显示： GATA-4可加强MSCs细胞对心肌梗死后缺氧心肌细胞的保护作用,使心肌梗死面积和心肌纤维化面积进一步降低。3. miR-195 通过与Bcl-w mRNA 的3′UTR 靶位点互补结合，抑制其翻译来降低Bcl-w蛋白水平。GATA-4抑制miR-195的表达，从而提高Bcl-w表达水平，由此加强MSCs对心肌梗死后缺血心肌的保护作用。上述研究表明：负调控miR-195能减少移植后MSC细胞凋亡，促进MSC细胞保护缺血心肌，。本课题拟研究负调控miR-195在体内体外抑制MSC细胞凋亡、增加存活率，促进MSC细胞保护缺血心肌。为MSCs细胞移植治疗心肌梗死提供新的支持。