内皮祖细胞外泌体介导miR-21及miR-200b促进血管新生修复大段骨缺损的作用及机制

The treatment of large segment bone defects is a clinical thorny problem. Recent studies have demonstrated that stem cell transplantation therapy promotes tissue injury healing mainly through the paracrine mechanism to stimulate the activity of receptor cells around the injury, rather than directly to certain parenchymal cells. Current reporting and our previous studies have indicated that exosomes have promise in tissue repair therapy as stem cell transplantation therapy by transmitting the biological active substance and information of the original cells, and the direct treatment with exosomes may overcome the limitations and risks associated with stem cell transplantation therapy, show a broad prospect on clinical application in the future. In consideration of our finding that exosomes derived from EPCs have obvious effect on promoting angiogenesis, the gene chip analysis suggests that exosomes derived from EPCs can activate the Erk1/2 pathway in HMECs, then we use microRNA array to show that exosomes derived from EPCs contain miRNAs (miR-21 and miR-200b) target Erk1/2 pathway. We use a series of experiments to clarify the new mechanisms that exosomes derived from EPCs contain functional miRNAs which can activate Erk1/2 pathway then promote bone regeneration by enhancing angiogenesis, to generate EPC-Exos with efficient enhanced function of angiogenesis.

大段骨缺损的修复一直是临床较为棘手的问题。研究显示干细胞在组织修复中发挥作用不是通过直接向特定实质细胞转化从而修复和替代受损组织，而是通过其旁分泌机制刺激损伤部位周围受体细胞活性。现有报道及我们的前期研究表明，外泌体可传递其来源细胞的生物活性物质和信息作用于效应细胞，从而发挥干细胞样生物学功能，并避免干细胞直接移植带来的潜在风险，显示出广阔的应用前景。我们在前期研究发现EPC来源外泌体可显著促进血管新生，基因芯片分析提示其可激活HMECs的Erk1/2信号通路，miRNA array分析提示其含有靶向此通路的关键miRNA, miR-21和miR-200b。据此，本课题拟通过系列实验，进一步明确EPC来源外泌体促进血管新生进而修复骨缺损的生物学功能，筛选并明确EPC来源外泌体中促进血管新生的关键miRNAs及其作用途径，构建具有高效促进血管新生的EPC-Exos，并阐明其修复骨缺损的机制。

大段骨缺损的修复一直是临床较为棘手的问题。本课题组长期致力于干细胞来源外泌体在组织损伤修复中的应用研究。本项目利用内皮组细胞（EPCs）来源外泌体结合生物材料修复大鼠大段骨缺损，通过生物信息学分析、荧光定量PCR、western blot和抑制剂阻断通路等研究手段系统的探讨EPC来源外泌体促进血管新生进而修复骨缺损的作用及可能的机制。研究结果表明EPCs来源外泌可能通过其内携带的miRNA126激活PI3K/AKT通路进而促进血管新生。随着研究的进展，我们同时发现骨缺损周围的炎症因子会促使细胞发生凋亡，严重影响修复效果，而外泌体具有明显抑制凋亡的作用。通过系列体内外实验课题组发现间充质干细胞来源外泌体通过携带的miRNA410抑制NLRP3炎症小体引起的细胞凋亡。研究结果揭示了外泌体在组织损伤中的修复作用，并进一步阐明了其作用机制，这将有助于对骨缺损修复调控机制的全面、深入的理解，并为骨缺损修复寻找新的治疗靶点提供理论依据。