骨髓间充质干细胞分泌exosomal miRNAs促进心肌细胞缺氧条件下存活的机制研究

A paracrine effect was regarded as the key mechanism involved in the mesenchymal stem cell-based treatment for myocardial infarction; and exosomes are one of several groups of secreted vesicles, possessing a great number of biophysical and biochemical propterties, which contribute to intercellular communication. However, whether MSC exerts effects on cardiomyocytes, through exosome secretion, has not been clearly elucidated yet. In our pilot experiments, hypoxia remarkably promotes MSC to paracrine exosomal miR-210 and miR-744，which, after being extracted from the supernatant, could significantly enhance the cardiomyocytes survival in hypoxic incubation, suggesting that exosomal miR-210 and miR-744 played critical roles in the favorable paracrine effect of MSC on cardiomyocytes. In this study, we will, using immunofluorescent co-localization and co-immunoprecipitation, further investigate the various phases of exosomal miRNA-210/744 behavior, including the paracrine from MSC, the uptake by cardiomyocyte, as well as the regulation of targeted mRNA and targeted pathways in cardiomyocyte. Furthermore, a myocardial infarction model of rat will be established to respectively confirm the key roles of exosomal miR-210/744 in the communication between MSC and cardiomyocytes, and then, in the beneficial effect of MSC on the improvement of ejection function after myocardial infarction. This study, delivering the novel insight to the exosomal miRNAs in the research of ischemic heart diseases, is expected to provide an experimental evidence for translational medicine in the promotion of clinical efficiency of cell-based treatment for cardiovascular diseases.

分泌作用是间充质干细胞（MSC）移植治疗心肌梗死的关键机制，而exosome是由细胞分泌后传递胞间信息的重要小体，但MSC是否可分泌exosome进入心肌细胞并发挥调控作用有待明确。本课题组前期发现缺氧培养明显促进MSC外泌exosomal miR-210和miR-744，提取的该活性成分可显著提高心肌细胞在缺氧条件下存活，因而推测其是介导MSC分泌作用保护心肌的重要分子。本研究将采用荧光共定位、动态活细胞成像、免疫共沉淀等技术分步骤阐明exosomal miR-210/744从MSC分泌、胞吞进入缺氧心肌、调控心肌内靶基因和靶通路等各效应阶段的现象及机制；并通过动物模型验证exosomal miR-210/744在MSC向心肌细胞传递促活信息、进而改善心功能的作用。本项目创新性地将exosomal miRNAs引入MSC治疗心肌梗死的机制研究，有望为提高干细胞疗效的临床转化提供实验依据。

本课题组严格按照原实验计划，针对“骨髓间充质干细胞分泌exosomal miRNAs促进心肌细胞缺氧条件下存活的机制研究“展开研究与探索。首先，通过探索MSCs分泌的exosomal miR-210对缺氧心肌细胞存活能力的影响，TUNEL及WB表现出exosomal miR-210对心肌细胞在缺氧条件下有保护作用。随后，通过构建慢病毒载体，转染MSCs-miR-210，观察到miR-210与exosome特异表面标志物的共定位，通过连续动态活细胞成像技术观测到exosome被心肌细胞的摄取过程。随后，我们探究被胞吞的exosomal miR-210在心肌内的生物学调控活性，结果显示心肌细胞的exosomal miR-210对靶基因（AIFM3）和靶通路（PI3K/Akt和p53通路）具有调控作用；直接在心肌细胞内过表达miR-210，同样验证miR-210对靶基因和靶通路的调控作用，结果发现摄入的exosomal miR-210与内生性的调控活性一致。最后，本课题组在体验证exosomal miR-210介导干细胞治疗心肌梗死的作用机制，实验结果显示其对心肌梗死动物的心功能保护作用，为临床改善干细胞治疗实际疗效提供理论基础。