人脐带间充质干细胞微泡通过传递miRNA影响类风湿关节炎患者滑膜成纤维细胞功能及机制的研究

Synovial fibroblasts (SFs) play a key role in synovial hyperplasia, joint inflammation and cartilage erosion in patients with rheumatoid arthritis (RA).Studies have shown that mesenchymal stem cell (MSC) is a special kind of cell population with multiple differentiation potentials and immunoregulatory properties,which can regulate immune disorders and inhibit bone destruction in RA patients, but its origin, ethics and safety factors have limited its clinical application. Mesenchymal stem cell-derived microvesicles(MSC-MVs) have abilities to regulate immune system and repair tissue like MSCs. It is an emerging hotspot in the field of stem cells, while the related research in the field of rheumatism is scarce. Our previous studies found that human umbilical cord MSC-MVs (UCMSC-MVs) can improve the joint symptoms and imaging findings of CIA rats, inhibit synovial hyperplasia,downregulate some proinflammatory cytokines and chemokine levels, and the effects are not weaker than the mother cells.This study focuses on the effects of UCMSC-MVs on the proliferation, invasion, migration and secretion of cytokines of RASFs, and then reveals its molecular mechanism by dynamic tracking the process of UCMSC-MVs transfering miRNAs to SFs，validating it from tissues, cells, molecules and genes levels in CIA rats. This establishes theory basic for the targeted therapy in RA based on UCMSC-MVs and miRNA transfered by UCMSC-MVs.

滑膜成纤维细胞（SFs）在类风湿关节炎（RA）患者滑膜增生、关节炎症、软骨的侵蚀破坏中起关键作用。研究表明间充质干细胞（MSCs）具有多向分化潜能及免疫调节能力，可调节RA患者的免疫紊乱、抑制骨破坏，但其来源、伦理学及安全性限制了其临床应用。间充质干细胞微泡（MSC-MVs）可模拟母细胞功能，同时规避MSCs的局限性，是干细胞领域的新兴研究热点，而风湿病领域研究甚少。本课题组前期研究发现人脐带MSC-MVs（UCMSC-MVs）可改善CIA大鼠关节症状及影像学表现，抑制滑膜增生，下调部分促炎因子及趋化因子的水平，且作用不弱于母体细胞。本课题将研究UCMSC-MVs对RASFs增殖、侵袭、迁移以及分泌细胞因子能力的影响，探讨miRNA传递在UCMSC-MVs治疗RA中的作用，为UCMSC-MVs以及基于UCMSC-MVs为载体的miRNA靶向治疗在RA中的应用奠定理论基础。

滑膜成纤维细胞（SFs）在类风湿关节炎（RA）患者滑膜增生、关节炎症、软骨的侵蚀破坏中起关键作用。间充质干细胞（MSCs）具有多向分化潜能及免疫调节能力，可调节RA患者免疫紊乱、抑制骨破坏，但其来源、伦理学及安全性限制了其临床应用。间充质干细胞微泡（MSC-MVs）可模拟母细胞功能，同时规避MSCs的局限性，是干细胞领域的新兴研究热点，而风湿病领域研究甚少。本课题组前期研究发现人脐带MSC-MVs（UCMSC-MVs）可改善CIA大鼠关节症状及影像学表现，抑制滑膜增生，下调部分促炎因子及趋化因子的水平，且作用不弱于母体细胞。本课题研究在体外实验部分表明hUCMSC-MVs抑制RASFs的增殖、迁移和侵袭能力，促进RASFs凋亡，对RASFs分泌细胞因子影响不显著；通过示踪UCMSC-MVs中的RNA明确UCMSC-MVs可以将RNA传递至RASFs；对miRNA作用验证时，通过敲除hUCMSC中Ago2抑制miRNA作用获得hUCMSCKD-Ago2-MVs，发现hUCMSCKD-Ago2-MVs可显著抑制RASFs增殖、迁移，但作用弱于hUCMSCNC-MV，证明miRNA传递在hUCMSC-MVs对RASFs生物学性状影响中发挥关键作用。Small-RNA测序检测到13个可能经由hUCMSC-MVs传递至RASFs中的miRNAs，并扩大样本量加以验证hUCMSC-MVs对上述miRNAs在UCMSCs和RASFs间传递的调节作用，其中miR-146a变化差异最大，miR-451a次之，因此hUCMSCs-Exo可能通过传递miR-451a至RASFs；将miR-451a负载于hUCMSC-MVs中（hUCMSC-MVsmiR-451a）进行体内外实验验证，miR-451a通过hUCMSC-MVs递送至RASFs，调控ATF2抑制RASFs的增殖、迁移和侵袭；在RA动物模型CIA大鼠踝关节中注射hUCMSCs-MVsmiR-451a通过miR-451a下调ATF2改善CIA大鼠关节症状及影像学表现；在体内外实验中hUCMSCs-MVsmiR-451a相较hUCMSCs-MVs显示出更好的治疗疗效。该课题首次确定miR-451a作为UCMSC-MVs靶向治疗RA的潜在miRNA靶点，为基于UCMSC-MVs为载体的miRNA靶向治疗在RA中的应用奠定理论基础。