间充质干细胞源性微粒通过lncRNA MALAT1/miRNA-21抑制肺微血栓形成减轻CPB后急性肺损伤

CPB-induced ALI has high mortality and is lack of effective treatment. Pulmonary microthrombus plays the key role during the CPB-induced ALI development. Research shows MSCs-MV is the potential therapy of ALI, but the mechanism is still unclear. Our previous experiments demonstrate that MSCs-MV can relieve CPB-induced ALI, which is the result of miRNA-21 overexpression in the lung, by transporting competitive endogenous RNA. We put forward the hypothesis that MSCs-MV transports lncRNA MALAT1 to pulmonary vascular endothelial cells, downregulating miRNA-21, inhibiting platelet activation, and relieving pulmonary microthrombus. The present study will explore how MSCs-MV preconditioning relieve CPB-induced ALI through RNA interference technique in both CPB-induced ALI model in rats and oxygen glucose deprivation model of human pulmonary vascular endothelial cells. The objective of this subject is to explicit how MSCs-MV protects lung from CPB-induced injury by modulating MALAT1/miRNA-21/ADAMTS18 axis. If success, the present study will unveil the therapeutic mechanism of MSCs-MV and give a new vision dealing with CPB-induced ALI.

体外循环（CPB）后急性肺损伤病死率高且缺乏有效治疗手段。肺微血栓形成是CPB后急性肺损伤发生发展的关键环节。研究表明间充质干细胞源性微粒（MSCs-MV）是急性肺损伤的潜在治疗手段，但机制未明。预实验发现MSCs-MV抑制CPB后肺微血栓形成，且与其携带的lncRNA MALAT1相关；肺血管内皮细胞miRNA-21表达升高诱发肺微血栓形成。生物学预测MALAT1是miRNA-21的竞争性内源性RNA。由此提出假说：MSCs-MV转运MALAT1至肺血管内皮细胞，下调miRNA-21，抑制血小板活化后肺微血栓形成，减轻CPB后急性肺损伤。本课题拟通过大鼠CPB和肺血管内皮细胞氧糖剥夺模型，用RNA干扰等技术探讨MSCs-MV减轻肺微血栓形成机制，阐明MALAT1对miRNA-21的调控，明确MSCs-MV治疗CPB后急性肺损伤的信号通路，有望为防治CPB后急性肺损伤提供新靶点和有效途径。

本课题从急性肺损伤这个亟待解决的临床问题出发，结合近年来的研究热点，从长链非编码RNA的角度入手，探索急性肺损伤的发生机制。本课题组发现在急性肺损伤患者中lncRNA MALAT1水平升高，小RNA miR-181a-5p降低，且两者呈负相关关系；凋亡因子Fas升高，炎症因子TNF-α，IL-1β和IL-6增加。课题组建立脂多糖LPS诱导的缺氧/复氧细胞模型，在人肺微血管内皮细胞HPMEC中通过mimic miR-181a-5p上调miR-181a-5p表达后，Fas基因表达降低，炎症因子表达减少，细胞凋亡比例减轻；而使用MALAT1的反义核苷酸si-MALAT1抑制其表达后，Fas基因表达同样降低，炎症因子表达减少，细胞凋亡比例减轻。通过双荧光素酶报告基因实验，结合生物信息学比对发现，miR-181a-5p是MALAT1的下游靶基因，Fas是miR-181a-5p的下游靶基因。提示MALAT1通过miR-181a-5p/Fas通路加重了急性肺损伤，加剧了炎症反应，增加了内皮细胞凋亡，从而促进了急性肺损伤的发生。本课题组在大鼠动物实验中再现了上述机制。本课题从表观遗传学和凋亡角度阐明了急性肺损伤的发病机制，为找到干预靶点提供了实验依据。