骨髓干细胞外泌体介导miR-124靶向整合素α2β1改善硬皮病皮肤纤维化的机制研究

Systemic scleroderma is an autoimmune disease that can cause fibrosis of the skin and internal organs, and has done a great harm to human health. Stem cell transplantation can alleviate skin fibrosis of scleroderma, but the specific mechanism remains unknown. Inflammation and epithelial mesenchymal transition (EMT) are important causes of skin fibrosis. Integrin α2β1 can regulate inflammatory response and EMT, which is a new target for inhibiting skin fibrosis. The latest investigations showed that the regulation of signal molecules by stem cell exosomes is an important mechanism for cell therapy. As a result, we speculate that adipose stem cells are likely to relieve inflammatory response and EMT by inhibiting integrin α2β1 through the exocrine, and inhibit the skin fibrosis. Our previous biological information analysis showed that miR-124 suppressed the expression of integrin α2β1, and also found that miR-124 expression in scleroderma patients was down-regulated. Therefore, upregulation of miR-124 can enhance the function of exosomes to resist fibrosis. This project aims to construct the overexpression/ interference miR-124 vector to transfect bone marrow-derived mesenchymal stem cells, and to observe the curative effect of stem cell exosomes to scleroderma in vivo and in vitro, and to clarify the relationship between molecular mechanism of cell therapy and fibroblast integrin α2β1 signaling pathway, to explore the integrin α2β1 downstream signal molecules, which will provide a new theoretical basis for the treatment of scleroderma.

系统性硬皮病可导致皮肤和内脏器官纤维化，严重影响人类健康。我们已证实骨髓间充质干细胞移植改善硬皮病皮肤纤维化，但机制不清。炎症和上皮-间质转化(EMT)是导致皮肤纤维化的重要原因。整合素α2β1可以调控炎症反应和EMT，是抑制皮肤纤维化的新靶点。最新研究显示，干细胞外泌体调控信号分子是细胞治疗的重要机制。因此我们推测，干细胞很可能通过外泌体抑制整合素α2β1改善炎症反应和EMT，抑制皮肤纤维化。我们前期经生物信息学分析发现，miR-124靶向抑制整合α2β1表达，还发现硬皮病患者miR-124表达下调，故上调miR-124可增强外泌体抗纤维化功能。本项目拟构建过表达/干扰miR-124载体转染骨髓干细胞，在体内外观察干细胞外泌体对硬皮病的疗效，明确细胞治疗的分子机制与成纤维细胞整合素α2β1信号通路的关系，探讨整合素α2β1下游信号分子，为硬皮病的治疗提供新的理论依据。

背景：硬皮病是以器官纤维化、免疫异常和血管损害为特征的自身免疫性结缔组织疾病。外泌体是细胞分泌的纳米级的小囊泡，充满了多种蛋白质、microRNA和DNA，是一种细胞间的相互作用方式。并且作为一种无细胞移植方法，外泌体避免了被机体清除，且活性更稳定。.目的：探究人脐带间充质干细胞来源的外泌体（HUMSCs-Ex）对硬皮病小鼠模型的治疗作用，并探索M1/M2巨噬细胞平衡在硬皮病中发挥的作用。.方法：使用透射电镜、纳颗粒跟踪分析（NTA）和纳米流式术（NanoFCM）鉴定HUMSCs-Ex。采用HE染色、Masson染色、TGF-β1免疫组化染色、Col1α1 mRNA和Col3α1 mRNA评价HUMSCs-Ex对硬皮病小鼠的治疗作用。利用免疫组化染色分析上皮间质转化的三个标志性蛋白，即α-SMA、Vimentin和E-cadherin。利用ELISA、Western blot和免疫荧光染色评价M1/M2巨噬细胞平衡在硬皮病小鼠皮肤的变化。.结果：HUMSCs-Ex能够减轻硬皮病小鼠真皮厚度和胶原沉积，降低α-SMA、Vimentin和升高E-cadherin在硬皮病小鼠皮肤的表达水平，还能抑制M2巨噬细胞极化从而恢复M1/M2巨噬细胞平衡。.结论：HUMSCs-Ex能够缓解硬皮病的纤维化程度，并且抑制上皮间质转化和维持M1/M2巨噬细胞平衡可能是HUMSCs-Ex发挥抗纤维化作用的可能途径。