ARDS肺微血管内皮细胞微环境调控MSC旁分泌HGF的机制研究

Diffuse damage of pulmonary microvascular endothelial cell (EC) is the basic pathological changes of ARDS. Effective repair of injured EC is a fundamental treatment of ARDS, which may help to improve its prognosis. Mesenchymal stem cells ( MSC) have potential beneficial effects on EC and lung injury repair of ARDS via paracrine hepatocyte growth factor ( HGF), however, the HGF secreted by MSCs may vary in the different conditions and influenced by injured EC microenvironment, the detailed mechanism is unclear. According to our previous study, we found that the secreted AngⅡ of injured EC and EC-MSC interaction may promote MSC to secrete more HGF. To elucidate the mechanism of injured EC microenvironment in the regulation of HGF secretion of MSC, in this study, MSC will be stimulated with AngⅡ, and the receptor and its downstream pathway will be blocked respectively. The role of Ang Ⅱ in promoting the secretion of HGF and the possible intracellular signaling pathway will be observed. Secondly, the AT1R and Cx43 genes of EC will be down-regulated, the modified EC will be injured by LPS and cultured with MSC directly, we will observe the expression of AT1R related pathway protein and the production of HGF on MSC, and then to clarify how EC-MSC contact could transfer AT1R mRNA through gap junction, which affect MSC secretion of HGF, and revealed the mechanism of injured EC microenvironment regulating the HGF secretion of MSC. Finally, we will also clarify whether overexpression of AT1R gene may enhance the targeting therapeutic effects of MSC in restoration of pulmonary endothelium and lung injury of ARDS through in vitro and in vivo experiments. This study will provide new strategy to reduce the mortality of ARDS.

有效修复弥漫性损伤的肺微血管内皮细胞(EC)是从根本上防治ARDS并改善其预后的重要策略。MSC可旁分泌HGF靶向促进EC修复，但HGF分泌受损伤微环境影响，具体机制不明。申请人前期研究发现损伤EC可能通过分泌的AngⅡ和EC-MSC接触增强MSC分泌HGF。为阐明EC微环境调控MSC分泌HGF的机制，本项目首先用AngⅡ刺激MSC，分别阻断其受体和下游通路，明确AngⅡ对MSC分泌HGF的促进作用及可能的胞内信号通路；其次将EC的AT1R和Cx43基因下调，与MSC接触培养，观察MSC上AT1R通路蛋白的表达及HGF的产生，明确EC-MSC接触如何通过缝隙连接转移AT1RmRNA，进而影响MSC分泌HGF，揭示EC微环境调控MSC分泌HGF的机制；最后通过细胞和动物实验，验证AT1R高表达优化的MSC可更好地靶向修复ARDS肺内皮和肺损伤。项目研究成果可为降低ARDS病死率提供新思路。

肺微血管内皮细胞(EC)是急性呼吸窘迫综合征(ARDS)时损伤的主要靶点同时也是ARDS失控炎症反应的重要来源， EC功能障碍介导ARDS的发生发展。促进EC修复就可能逆转ARDS的病理生理进程，但缺乏有效治疗方法。近年来研究发现MSC可以通过旁分泌HGF促进ARDS损伤EC的修复，但其疗效有限且MSC旁分泌HGF受到损伤EC微环境的调控，体外细胞实验显示微环境对MSC产生的影响包括两方面，一方面来源于损伤EC分泌到胞外的因子，另一方面来源于损伤细胞本身与MSC之间的直接接触，具体机制均不清楚。我们前期研究发现损伤EC可能通过分泌的AngⅡ和EC-MSC接触增强MSC分泌HGF。 因此本研究通过体外细胞实验证实损伤EC分泌的AngⅡ和EC-MSC直接接触对MSC分泌HGF的促进作用并探讨其具体的信号分子机制。并在此基础上优化MSC以增强MSC分泌HGF的能力，并通过体外细胞实验及动物实验证实优化后的MSC具有更强的修复损伤内皮及肺组织的作用。. 本研究的主要创新贡献包括：1) 证实了损伤 EC通过分泌的Ang Ⅱ激活MSC的AT1R-PI3K/AKTNF-κB通路促进MSC旁分泌HGF；2) 阐明损伤EC通过细胞间Cx43缝隙连接向MSC转移AT1R mRNA，进一步促进MSC旁分泌HGF；从而明确了损伤EC微环境对MSC旁分泌HGF的影响机制。3) 建立了高表达AT1R的MSC株, 并证实其在体外可促进LPS损伤EC的修复, 改善内皮功能。4) 体内动物实验进一步证实高表达AT1R的MSC移植可靶向增加肺组织内HGF的表达，更好的修复ARDS肺内皮及肺组织。. 本研究通过研究ARDS损伤组织与MSC的交互作用，探讨了ARDS损伤EC微环境对MSC旁分泌HGF的影响及具体机制，并成功构建了高表达AT1R的MSC细胞株，优化MSC功能，增强MSC靶向修复ARDS肺内皮及肺损伤的效果，提高MSC靶向治疗ARDS的疗效。为ARDS肺损伤的修复开辟了干细胞治疗的新途径。.