基于RhoA/ROCK通路研究间充质干细胞外泌体-miRNA对急性肺损伤肺血管内皮通透性的调控作用

Acute lung injury (ALI) is a clinical syndrome with extremely high mortality. The pathogenesis is complicated and the treatment is difficult. The change of pulmonary vascular endothelium permeability may be one of the main pathogenesis. Studies have shown that microRNA（miRNA） or mesenchymal stem cell (MSC)exosome can regulate vascular endothelial permeability of ALI. We have previously demonstrated that human placental MSCs can relieve ALI in mice and improve pulmonary vascular endothelial permeability; miRNAs associated with ALI in MSC-exosome have been isolated, identified, and screened. We will further screen out MSC-exosome-miRNA related to vascular endothelial permeability of ALI by bioinformatics technology; construct miRNA interference or overexpression lentivirus to verify the effect of exosome-miRNA for vascular endothelial permeability in ALI; The RhoA/ROCK knockdown plasmid was used to detect and analyze the expression of RhoA/ROCK and its upstream or downstream molecules. The regulatory relationship between MSCs secreted by paracrine exosome-miRNA and RhoA/ROCK signaling pathway was explored. The implementation of this project will partially clarify the molecular mechanism of MSC-exosome-miRNA for the regulation of pulmonary vascular endothelial permeability and alleviation of ALI, and provide theoretical basis for MSC cell-free treatment of ALI.

急性肺损伤（ALI）是死亡率极高的临床综合征，发病机制复杂、治疗困难，肺血管内皮通透性改变可能是其主要发病机制之一。研究表明miRNA或间充质干细胞（MSC)外泌体均可调控ALI肺血管内皮通透性。前期我们已证实人胎盘MSC可缓解小鼠ALI、改善肺血管内皮通透性；分离、鉴定并筛选了MSC外泌体中与ALI相关的miRNA。我们将通过生物信息学技术进一步筛选出与ALI肺血管内皮通透性相关的MSC外泌体-miRNA；构建miRNA干扰或过表达慢病毒，验证外泌体-miRNA对ALI肺血管内皮通透性的影响；通过RhoA/ROCK敲降质粒，检测并分析RhoA/ROCK及其上下游分子表达，探明MSC旁分泌的外泌体-miRNA与RhoA/ROCK信号通路存在的调控关系。本项目实施将部分阐明MSC外泌体-miRNA调节肺血管内皮通透性缓解ALI的分子机制，为MSC无细胞治疗ALI提出理论依据。

急性肺损伤（ALI）是死亡率极高的临床综合征，发病机制复杂、治疗困难，肺血管内皮通透性改变可能是其主要发病机制之一。研究表明miRNA或间充质干细胞（MSC)外泌体均可调控ALI肺血管内皮通透性。本研究通过分离培养人胎盘MSC，收集其培养上清，从中提取MSC-MV，在ALI体内体外模型探究人胎盘MSC或MSC-MV对肺血管内皮通透性的影响。随后利用高通量测序及生物学分析进一步寻找人胎盘MSC-MV中与HPVEC通透性相关的miRNA及miRNA靶基因，明确miRNA及RhoA/ROCK等相关信号通路对肺血管内皮通透性的影响。实验成功分离及获取人胎盘MSC，通过表面特异性抗原检测及三系诱导分化鉴定成功后提取其MSC-MV，并利用透射电镜及Western blot技术进行鉴定。体外实验证实人胎盘MSC及其外泌体能有效减轻LPS诱导的ALI小鼠病理损伤及通透性，体内实验证实人胎盘MSC外泌体可以改善LPS损伤后的HPVECs血管生成、迁移、细胞骨架及凋亡等功能。进一步通过高通量测序分析发现MSC-MV体内富含多种miRNA，其功能主要富集在细胞连接、血管生成、炎症和能量代谢的信号通路中，包括613个已知和712个未知的miRNA，以hsa-miR-148a-3p表达丰度最高，生物信息学分析确定ROCK1是hsa-miR-148a-3p的靶点。Q-PCR表明hsa-miR-148a-3p与细胞损伤显著相关。通过构建富含hsa-miR-148a-3p的MSC-MV，从而研究其对ALI肺血管内皮通透性的影响，结果显示过表达hsa-miR-148a-3p的外泌体能够更好促进ALI体外模型HPVEC的迁移、血管生成等细胞功能的恢复，并能够有效降低了ROCK1表达水平，进一步构建过表达ROCK1的HPVEC，并用MSC-MV进行干预，发现其治疗效果降低，结果表明hsa-miR-148a-3p/ROCK1途径可能是人胎盘MSC-MV改善HPVEC功能的重要途径。本项目的实施对于探索以MSC-MV为基础，开展特异、高疗效无细胞治疗ALI 和其他疾病的临床前研究具有一定的理论价值和实践意义。