整合素α2β1在间充质干细胞外泌体靶向创面微环境及其抑制增生性瘢痕的作用机制研究

The pathology of hypertrophic scar (HTS) mainly contains over-differentiation of fibroblast (Fb) to myofibroblast (mFb), and collagen deposition in extracellular matrix (ECM). Previous studies have established the regulation of Fb differentiation and inhibition of HTS by mesenchymal stem cell-derived exosomes (MSC-Exo) via miRNA delivery. On top of such mechanism, we have further found that MSC-Exo locates wound microenvironment and adheres to Fb-derived ECM, while the key components and mechanism of its targeted signal transduction remains for further investigation. By means of isobaric tags for relative and absolute quantitation (iTRAQ) and bioanalysis, we have then illustrated the key role of integrin α2β1（ITGα2β1）in this procedure. Our research proposal thus aims to: (1). Study the effect of ITGα2β1-mediated exosomes on Fb differentiation and targeted aggregation of Fb-derived ECM; (2). Identify the ligand(s) and adhesion mechanism of ITGα2β1 by using integrin-mutated exosome and co-IP; (3). Examine the prevention of HTS by our “ITGα2β1-exosome-wound microenvironment” approaches in animal models. Overall, this study will not only elucidate the mechanism of mFb over-differentiation targetedly suppressed by MSC-Exo, but also provide evidence for an efficient molecular therapy of HTS, as well as shed light on future research in relevant fields.

成纤维细胞（Fb）向肌成纤维细胞（mFb）过度分化和胶原在细胞外基质（ECM）大量沉积是增生性瘢痕（HTS）的主要病理基础。研究证实，间充质干细胞来源的外泌体（MSC-Exo）可通过向Fb转运miRNA调节其分化并抑制瘢痕增生。在此基础上我们发现MSC-Exo具有定向创面微环境和黏附Fb周围基质的现象，而参与这一靶向信息传递的物质和机制仍待进一步研究。利用iTRAQ及生物学分析我们发现整合素ITGα2β1可能起到关键作用。本课题拟研究ITGα2β1介导外泌体对Fb周围基质靶向聚集作用和对Fb分化功能的影响；利用整合素突变外泌体、免疫共沉淀等技术确定ITGα2β1相应配体及粘附机制；最终通过动物模型检验“ITGα2β1-Exo-创面微环境”对mFb靶向抑制调控及预防HTS的作用。本研究不仅可阐明MSC-Exo靶向调控mFb过度分化作用机制，更为HTS分子防治及效率优化提供新的理论依据和方向。

抑制肌成纤维细胞过度形成和细胞外基质中胶原蛋白的过度分泌，是目前创面修复及瘢痕研究领域的热点问题，也是预防增生性瘢痕发生的关键靶点。然而如何能够高效促进创面愈合同时抑制瘢痕形成是一个难点问题。随着外泌体研究的不断深入，对于外泌体功能的认识从囊泡内含物，逐渐扩展至囊壁的多种膜蛋白结构。我们研究发现脐带间充质干细胞来源的外泌体（uMSC-Exo）能够通过体循环富集于受损的创面环境并选择性聚集于成纤维细胞（Fb）周围基质并且参与创面修复的整个过程。利用同位素标记相对和绝对定量（iTRAQ）技术首次完成了uMSC-Exo蛋白定量分析。通过比对发现uMSC-Exo中包含多种膜蛋白，其中整合素ITGα2β1丰度突出，微颗粒细胞流式和Western Blot 数据也进一步验证了这个结果。体外实验证实了ITGα2β1介导的uMSC-Exo向Fb周围ECM的靶向聚集作用并检测其对Fb分化功能的具体抑制影响。并且通过黏附实验检测不同胶原及FN对ITGα2β1的亲和情况，以确定ITGα2β1所对应的配体——新生胶原。进一步运用免疫共沉淀技术验证ITGα2β1与特异性配体结合情况。最终通过兔耳模型验证ITGα2β1介导的uMSC-Exo靶向转运机制在抑制增生性瘢痕中的作用即uMSC-Exo通过膜蛋白ITGα2β1对成纤维细胞周围ECM中特定配体蛋白选择性黏附，进而被成纤维细胞特异性摄取，抑制其过度分化和胶原分泌，改善创面微环境，最终实现抑制瘢痕增生。本课题从全新的角度丰富了MSCs 在创面修复领域的相关机制研究，并为增生性瘢痕分子治疗的效率优化提供新的理论依据和研究靶点。