基于蜕膜巨噬细胞外泌体miRNA调控TLR-4/NF-κB信号通路探讨补肾活血法干预URSA的分子机制

母胎界面免疫紊乱诱发微血栓形成与不明原因复发性流产（URSA）密切相关。课题组前期采用补肾活血法获得可靠临床疗效，并发现药物可能是通过TLR-4/NF-κB信号通路减少血管内皮细胞（EC）损伤而发挥作用，但机制不清；近期研究发现蜕膜巨噬细胞(DM)外泌体参与局部免疫调节，其携带的miRNA可调控EC功能。由此推测：补肾活血法可能干预了DM外泌体传递的miRNA，通过TLR-4/NF-κB通路调控EC而减少血栓形成。采用CBA/J-DBA/2孕鼠流产模型，用经验方丹寿汤干预后高通量测序DM外泌体miRNA表达谱，差异分析及靶基因预测和通路富集分析；用Transwell建立EC与DM共培养体系，设外泌体拮抗剂与药物血清干预对照，观察DM分泌和EC摄取外泌体能力、TLR-4/NF-κB信号通路关键分子mRNA及前期筛查的高差异表达miRNA水平，探索中药干预母胎界面DM外泌体miRNA调控机制。

Microthrombus formation induced by maternal-fetal interface immune disorder is closely related to unexplained recurrent spontaneous abortion (URSA) . In the early stage, the research group adopted kidney-tonifying and blood-activating method to obtain reliable clinical efficacy. It was also found that the drug may act through the endothelial TLR-4 /NF-κB signal pathway. But the mechanism is unclear. Recent studies have found that decidual macrophages (DM) exosomes participate in local immune regulation, the miRNA it carries regulates the function of endothelial cells. We speculated that the method of kidney tonifying and blood activating may interfere with DM exosomes and the miRNA transmitted. Endothelial cells are regulated by the TLR-4 /NF- B pathway to reduce thrombosi. CBA/J-DBA /2 mice abortion model was adopted. The differential expression profile of miRNA in DM exosomes was detected by high-throughput sequencing after the intervention of empirical Danshou decoction. Bioinformatics method was used for target gene prediction and pathway enrichment analysis. The co-culture system of EC and DM was established by Transwell methods, and the exosome antagonist group and drug serum intervention control group were set up. The ability of endothelial cells absorbing exosomes, the mRNA levels of key molecules of TLR-4 /NF- κB signaling pathway and the high differential expression of miRNA in the previous screening were detected. The purpose of these is to analyze the miRNA regulation mechanism of TCM intervention on maternal-fetal exosomes.