外泌体介导miRNA127导入系统的建立及其调控脓毒症急性肺损伤作用研究

Sepsis induced acute lung injury (ALI) presents as acute respiratory distress syndrome which is caused by the release and activation of a large number of inflammatory mediators and inflammatory cytokines during sepsis, leading to damage of alveolar epithelial cells and capillary endothelial cells, resulting in diffuse pulmonary interstitial and alveolar edema. Mesenchymal stem cell-derived exosomes (MSC-exosomes) are mesenchymal vesicles treleased from resting or activated mesenchymal stem cells into the extracellular matrix. Proteins and RNA can be well protected by the membrane structure of exosomes so as to successfully reach target cells and exert their protective effect on tissues and organs. It was found that miRNA-127 could reduce the expression of CD64 in alveolar macrophages and reduce inflammation in the lungs, but the expression of endogenous miRNA-127 was lower, while MSC-exosomes could play a protective role in the lungs of sepsis induced ALI. This project is to construct a system for the induction of miRNA-127 into the rat model of septic ALI mice, and to observe its effects on survival rate, lung structure and function, CD64 expression of alveolar macrophages through miRNA-127-CD64 signal axis. We propose to elucidate the protective mechanism of miRNA-127 loaded MSC-exosomes on sepsis induced ALI and to provide a new way for clinical treatment of sepsis induced ALI.

脓毒症急性肺损伤（ALI）是脓毒症时大量炎症介质及细胞因子释放和活化，导致的肺泡上皮细胞及毛细血管内皮细胞损伤，形成弥漫性肺间质及肺泡水肿，临床表现为急性呼吸窘迫综合征。间充质干细胞源性外泌体（MSC-exosomes）是MSCs在静息或活化状态下释放到细胞外基质的膜性小囊泡，其携带的RNA等能被自身膜结构较好保护，顺利到达靶细胞。研究发现MSC-exosomes能够对脓毒症ALI发挥肺保护作用、miRNA-127可降低肺泡巨噬细胞内CD64表达，减轻肺部炎症，但内源性miRNA-127表达较低。本项目拟构建外泌体介导miRNA127导入系统，作用于脓毒症ALI小鼠模型，通过miRNA127-CD64信号轴观察其对小鼠生存率、肺结构功能、肺泡巨噬细胞CD64表达等的影响，阐明负载miRNA-127的MSC-exosomes对脓毒症ALI的肺保护机制，旨在为临床治疗脓毒症ALI提供新思路。

背景:中性粒细胞细胞外陷阱(NETs)在脓毒症相关急性肺损伤(ALI)中起重要作用。BMSCs来源的外泌体和miRNA正在成为治疗ALI的有前途的药物。本研究旨在阐明骨髓间充质干细胞(BMSCs)来源的外泌体携带miR-127-5p抑制脓毒症相关ALI中NETs形成的机制。.方法:利用小鼠骨髓间充质干细胞上清分离外泌体，利用qRT-PCR检测miR-127-5p在外泌体中的相对表达量。利用miR-127-5p抑制剂下调BMSCs来源的外泌体中的miR-127-5p水平。气管内注射脂多糖(Lipopolysaccharides, LPS)建立ALI小鼠模型，用不同的外泌体或联合CD64单克隆抗体进行处理。采用HE染色、组织损伤评分及湿/干(W/D)重比评价肺组织病理变化。ELISA法检测TNF-α、IL-6、IL-1β浓度，流式细胞术检测外周血中性粒细胞数量。采用免疫荧光、Western blotting和ELISA检测与NETs相关的蛋白(包括MPO、NE和H3)。最后，双荧光素酶报告基因实验证实了miR-127-5p与CD64的相互作用。.结果:我们成功地从BMSCs中分离出外泌体，并证实了miR-127-5p会在外泌体中富集。与LPS组和LPS+ exo抑制剂组相比，BMSCs来源的外泌体处理的ALI小鼠组织学改善。同时，LPS+EXO-NC组小鼠肺组织和外周血中NETs及炎症因子释放量较LPS组和LPS+ exo抑制剂组降低。通过双荧光素酶报告基因检测，我们确定CD64是miR-127-5p的直接靶点。同时，EXO-NC可与抗cd64单抗在ALI小鼠中协同作用，减轻组织损伤，抑制炎症因子释放和NETs形成。当miR-127-5p下调时，外泌体的协同作用减弱。.结论:研究结果表明，来自BMSCs的外泌体miR-127-5p是一种潜在的治疗脓毒症诱导的ALI的药物，它通过靶向CD64减少NETs的形成。