内皮祖细胞源性微粒microRNA抑制内皮间质转化改善心脏纤维化的机制研究

Increasing studies observed that EndMT played pivotal role in the progression of cardiac fibrosis(CF). Previous study found that endothelial progenitor cell (EPC)-derived microvesicles(MV) could inhibit CF by transporting protein and RNA. Our data also indicated that EPC-MV could inhibit CF in transverse aortic constriction (TAC) model. However, the underlying mechanism was unclear. Hence, our project aimed to explore the role of EndMT in EPC-MV mediating CF inhibition. EPC was stimulated with the serum from sham-operation mice and TAC-induced heart failure mice, and then EPC-MV were collected respectively(sham-EPC-MV and hf-EPC-MV). The data showed that sham-EPC-MV had a more potent effect in suppressing TGF-β induced EndMT, which could be abolished by pretreating EPC-MV with RNAse. These data indicated that RNA containing in EPC-MV might mediate EndMT inhibition. Furthermore, miRNA array between two types of MV was done. The data showed that fifteen miRNA were significant higher in sham-EPC-MV than in hf-EPC-MV. Hence, this project aimed to screen out the key miRNA which mediated the effect of EPC-MV in suppressing EndMT. Moreover, the target gene of miRNA was explored. This project would help to explain the therapeutic mechanism of EPC-MV in CF and also highlight the role of EndMT in this issue.

心脏纤维化（CF）是心力衰竭领域的研究热点，新近认为内皮间质转化（EndMT）是其重要途径。有报道内皮祖细胞（EPC）可通过分泌微粒传递蛋白和RNA而抑制纤维化。我们也发现EPC微粒可减轻主动脉缩窄（TAC）诱导的CF及心脏EndMT水平，但其机制尚不清楚。此外，我们通过用假手术及心衰小鼠血清孵育的EPC其微粒处理内皮细胞，发现前者抑制EndMT作用明显强于后者，以RNA酶处理微粒后抑制作用均消失，说明EPC微粒主要通过RNA起作用。对EPC微粒行miRNA芯片发现15个miRNA在假手术血清孵育的EPC微粒中表达明显高于心衰血清孵育的EPC微粒，提示EPC 微粒通过miRNA抑制EndMT。故本项目拟通过体内TAC及体外TGF-β诱导的EndMT模型，在候选miRNA中筛选出目标miRNA，验证它在EPC微粒抑制EndMT中的作用并寻找其靶基因，为干预EndMT途径实现抗CF提供新思路。

心脏成纤维细胞是心室重构和心脏纤维化中最主要的效应细胞之一。心脏成维细胞除了源于原位成纤维细胞的自身增殖外，内皮间质转化（endothelial-to-mesenchymal transition, EndMT）是心脏成纤维细胞的另一来源。项目按照原计划执行，已基本完成所有研究内容。通过体内TAC及体外TGF-β诱导的EndMT模型，在候选miRNA中筛选出目标miRNA，验证了它在EPC外泌体抑制EndMT中的作用。在另一方面，心脏原位的成纤维细胞也是心脏纤维化的主要效应细胞。我们在原项目基础上进一步探讨了EPC外泌体对成纤维细胞活化的作用。结果发现，EPC源性外泌体通过miR-10b-5p靶向干预SMURF1和HDAC4蛋白，从而抑制了TGF-β1信号通路依赖的成纤维细胞活化。并且，该作用只在常氧培养的EPC外泌体中存在，在缺氧培养的EPC外泌体中明显减弱。该部分研究内容，从原位成纤维细胞的角度更全面地评估EPC外泌体对心脏纤维化的影响，是对原项目很好的补充。在该项目资助下，已累计发表论著3篇，其中第一作者发表SCI 论著2篇（总IF=6.36），部分数据待发表。项目的科学意义在于（1）发现EPCs外泌体通过抑制心脏EndMT以及成纤维细胞活化两条途径，减轻心衰时心脏纤维化。为将来进一步利用EPC外泌体作为干预手段用于心脏纤维化的治疗提供依据。（2）揭示了EPC外泌体抑制心脏纤维化的可能机制，明确了外泌体中起重要作用的miRNA，为疾病的干预提供了新靶点。