MicroRNA-326在心脏干细胞移植治疗心肌梗死中的作用及机制研究

There are several problems such as poor engraftment, survival, and ability of differentiation in the transplantation of stem cells to treat myocardial infarction. In previous study, we found that the function of myocardial repair by cardiac stem cells was significantly improved after cardiac stem cells were pretreated with bone marrow mesenchymal stem cells derived Exosomes. Further, miR-326, one of the significant different genes, was screened. Recent study revealed that miR-326 could modulate the self-renewal and differentiation ability of cancer stem cell by inhibiting the Hedgehog signaling pathway. Yet, the relation among miR-326, Hedgehog pathway and the functions of myocardial repair by cardiac stem cells are still poorly understood. To this end, we put forward the hypothesis that miR-326, through inhibiting the Hedgehog pathway, reduced the ability of mobilization, differentiation, and myocardial repair of cardiac stem cells. To test this hypothesis, from the different level of molecules, cells to animal in vivo, we will study the regulation of myocardial repair functions by the miR-326 in the transplantation of stem cells to treat myocardial infarction, through the mice cardiac stem cells, the mice myocardial infarction model, using Western blot, lentivirus vector transfection and immunofluorescence, etc. And to demonstrate the mechanism of miR-326 signal transduction in the process of cardiac stem cells playing a myocardial repair function. This study will provide a new idea of stem cell therapy for patients with myocardial infarction.

心脏干细胞（CSCs）移植治疗心肌梗死存在着干细胞存活定植、分化等能力欠佳的难题。在前期研究中，我们发现CSCs经BMSCs来源的Exosomes刺激后，心肌修复功能显著提高；并筛选出差异表达显著的miR-326。最新发现，miR-326通过抑制Hedgehog信号通路，能够降低肿瘤干细胞自我更新及分化能力。迄今对miR-326及Hedgehog通路与CSCs功能的关系仍知之甚少。为此，我们提出假说：miR-326可能通过抑制Hedgehog通路，降低CSCs的动员、分化等功能。为了验证这一假说，我们将通过小鼠CSCs、小鼠心肌梗死模型，采用Western-blot、慢病毒载体转染、免疫荧光等手段，从分子、细胞及动物整体水平不同层面探讨miR-326对CSCs的功能调控作用，明确miR-326在CSCs发挥心肌修复功能中的信号调控机制。本研究将为心梗患者提供新的干细胞治疗思路。

干细胞移植治疗缺血性心脏病已成为全球一大研究热点，也是被期盼为治疗心肌梗死（MI）最有前途的研究方向。本团队成员曾证实，应用内皮祖细胞EPCs治疗MI，具有促新生血管形成、降低MI面积、改善心功能的能力。另外，在前期研究中我们发现miR-326-5p能够增强干细胞促血管形成能力。本项目中，研究miR-326-5p对EPCs血管形成能力的影响，以及在EPCs移植治疗MI中发挥的功能作用及相关机制。.首先应用密度梯度离心法提取小鼠骨髓中EPCs，倒置显微镜下观察细胞生长情况及形态，流式细胞仪检测细胞表面标记物，免疫荧光染色观察EPCs内吞ac-LDL。从多个方面鉴定EPCs。数据库查询，结合双荧光素酶报告实验、RT-qPCR、WB检测，明确miR-326-5p的靶基因为Wnt1。通过体外Tube formation实验，荧光观察DiI-EPCs嵌插整合进入tube的数目；以及进一步通过体内皮下基质胶实验，免疫荧光染色CD31观察形成的血管数目，研究miR-326-5p对EPCs促血管形成能力的影响及其机制（分组：NC、miR-326-5p agomir、miR-326-5p antagomir、miR-326-5p agomir+Wnt1 agonist）。研究结果表明，miR-326-5p通过调节靶基因Wnt1，能够显著增强EPCs促血管形成能力。建立小鼠心肌梗死模型，EPCs移植治疗MI，通过心脏彩超检测、Masson’s Trichrome染色分析观察对心功能、心肌纤维化的影响；免疫荧光染色BS1 lectin及α-SMA，观察受损心肌毛细血管及小动脉发生情况。实验分组：Control、EPCs-NC、miR-326-5p-EPCs、miR-326-5p-EPCs+Wnt1 agonist。研究结果表明，miR-326-5p通过调节EPCs促血管形成能力，能够显著增强EPCs移植治疗MI疗效，包括改善心功能、降低心肌纤维化。.本项目研究，揭示miR-326-5p靶向作用于Wnt1，能够增强EPCs血管形成能力；证实miR-326-5p-EPCs移植治疗心肌梗死，可能通过促进受损心肌血管发生，来提高心肌修复能力，包括心功能的改善、心梗面积的降低。