生物工程化iPS-MSCs来源的外泌体修复缺血性组织损伤的作用及机制

Lacking of ideal clinical therapy methods have long been the main problems of ischemia diseases. Recent studies indicate that stem cell-derived exosome containing various bioactive molecules, t hese include several growth factors, mRNA and microRNA. Its positive functions in promoting angiogenesis in ischemic injury tissue after transplanted besides avoiding potential risks on stem cell transplanting show a broad prospect on clinical application in the future. Therefore, obtaining exosome that gather injury tissue efficient and exert restoring function becomes a key scientific problem that need to be solved. In consideration of our previous study that exosome derived from iPS-MSCs has obvious effect on promoting angiogenesis in ischemic hind limb, we use solex deep sequencing and a series of experiments in vivo and in vitro to screen functional miRNAs that have a notable role in angiogenesis；By using genetic engineering technology to generate iPS-MSCs that over-expression chemokine receptor CXCR4 and functional miRNAs，then to obtain exosome which can rally round injury tissue to promote angiogenesis efficiently，and clarify the molecular mechanisms on how exosome exert injury tissue repairing function. Our project aims to establish a new technology strategy on taking advantage of stem cell to restore ischemia-like injuries, which has significant practical sence on regenerative medicine.

缺血性疾病一直缺乏理想的临床治疗手段。最新研究显示干细胞分泌的外泌体含有多种生长因子、编码或非编码RNA等生物活性物质，移植到损伤组织可促进血管新生修复组织损伤，避免了干细胞直接移植带来的潜在风险，展现出广阔的临床应用前景。如何获得能高效聚集于损伤部位并发挥损伤修复作用的外泌体就成为亟待解决的关键科学问题。本项目在发现iPS-MSCs来源外泌体具有明显促血管新生修复下肢缺血性损伤的前期基础上，拟通过Solex深度测序技术及体内外实验，筛选出可显著促进血管新生的功能性miRNAs；利用干细胞趋化性迁移特性，通过基因工程技术，构建同时过表达趋化因子受体CXCR4和功能性miRNAs的iPS-MSCs，以获得既可向损伤部位定向聚集并能高效促血管新生修复损伤的外泌体，并探索其修复损伤的机制。项目的实施将建立利用干细胞修复缺血性等组织损伤的新技术策略，对干细胞再生医学技术的临床转化具有重要现实意义。

缺血性组织损伤是临床缺血性疾病中常见的病理过程，尽快恢复缺血组织的血液供给有助于各种缺血性损伤的修复。近年来，间充质干细胞来源外泌体（MSC-exosome，MSC-Exo）展现出较成体MSCs更强大的减轻缺血性组织损伤，促进缺血组织血管新生及功能恢复的功能。但是，由诱导多能干细胞分化形成的间充质干细胞（iPSCs-MSC, iMSCs）来源的外泌体（iMSCs-exosome, iMSC-Exo）是否具有强大的修复缺血性损伤的功能及其机制值得深入研究。本实验通过建立下肢缺血模型、脑缺血模型、皮肤缺损模型、颅骨缺损模型及股骨头坏死模型，系统评价了iMSC-Exo对缺血器官或组织的修复作用及其促进血管新生的分子机制。结果发现，iMSC-Exo可以修复缺血下肢和缺血脑组织，并促进缺血组织内血管新生，miRNAs芯片结果也证实， iMSC-Exo内含有32种血管新生相关的miRNAs，并且高丰度表达miR-210、 miR-126、 miR-103、miR-424、miR-378、let-7α、let-7e这7种促血管新生miRNAs。同时，动物实验也发现，iMSC-Exo可以促进皮肤缺损的修复和促进骨的再生。此外，iMSC-Exo通过激活PI3K/Akt信号通路增强内皮细胞的成血管能力而防治股骨头坏死的发生。为了提高外泌体的靶向治疗效率，我们对外泌体表面进行了RGD修饰，通过脑胶质瘤皮下模型发现外泌体能够高效率的靶向到肿瘤组织。本项研究了建立利用干细胞修复缺血性等组织损伤的新技术策略，对干细胞再生医学技术的临床转化具有重要现实意义。