内皮祖细胞源性外泌体促进MSCs修复骨缺损的研究及HMGB-1的作用机制

One of the reasons that limit the clinical application of MSC graft is insufficiency or deficiency of the vascular network. Based on the previous experimental results, EPCs in vitro contact culture with MSCs significantly increases MSC angiogenesis and osteogenesis, and EPCs exosomes and specific phenotypes were obtained, suggesting that EPC-derived exosomes should play a paracrine role through cytokine release. Further pre-experiment showed that HMGB-1 was expressed in both the cell body and the exosomes, and the migration and vasoactive activity of EPCs were enhanced after overexpression of HMGB-1 gene. Based on these results, we proposed the hypothesis that HMGB-1 may improve the biological activity of EPCs and MSCs through autocrine and paracrine by releasing exosomal cytokine release of EPCs, and promote angiogenesis and osteogenesis to facilitate bone defect repair. This project intends to observe the effect on cell migration, osteogenesis, vascular differentiation of EPCs and MSCs, that stimulated by EPC-derived exosomes. We further investigate the molecular mechanisms that EPC-derived exosomes may improve the potential biological activities of EPCs and MSCs and promote angiogenic and osteogenic effects, by releasing HMGB-1 to activate downstream signaling pathway. We also establish femoral bone defect model in rats, detect the angiogenesis and osteogenesis in vivo of combined application of MSCs and EPCs modified with overexpression of gene HMGB-1 in vivo repair test, to validate that HMGB-1 as a biological active factor may promote the repair of bone defect repair. Finally, we hope to provide the experimental basis for the clinical application that EPCs combined with MSCs are used as a new therapeutic strategy for the treatment of large segmental bone defect.

限制MSCs骨科临床应用的原因之一是移植物内血管网生成的不足或缺乏。前期工作发现内皮祖细胞（EPCs）体外共培养显著提高MSCs血管生成和成骨效应，并获得EPCs外泌体及鉴定表型，提示其可能通过外泌体发挥旁分泌作用；进一步预实验表明HMGB-1因子在胞体及外泌体均有明显表达，而且过表达HMGB-1后EPCs迁移、成血管活性增强。由此，我们提出HMGB-1可能是通过EPCs外泌体的释放，以自分泌、旁分泌的方式改善EPCs和MSCs的生物学活性，促进成血管、成骨效应以利于骨缺损修复的科学假设。本项目拟将HMGB-1过表达EPCs，观察其源性外泌体对EPCs和MSCs细胞迁移、成骨、成血管等活性的影响，探讨其可能通过释放HMGB-1启动信号通路产生生物效应的分子机制；并建立大鼠股骨缺损模型进行体内验证，为基因修饰的EPCs联合MSCs应用于骨缺损的修复作为一种新的治疗策略应用于临床提供实验依据。

严重牙周炎骨吸收、颌骨创伤、骨感染、颌骨肿瘤切除等原因所致的颌骨缺损十分常见，目前仍然是口腔临床所面临的一个难题。组织工程移植体修复骨缺损过程中，血管生成是最重要的步骤。最近研究表明高迁移率族蛋白1（HMGB-1）在调节ECs和MSCs 细胞生物学功能方面起重要作用。HMGB-1既能够增强ECs的归巢迁移、成血管的功能，又可以促进MSCs归巢迁移和成骨分化，而这些生物学行为都是干细胞修复骨缺损所必需的，也为ECs 和MSCs 的联合应用提供了有用的活性因子。为此，我们将MSCs过表达HMGB-1，观察HMGB-1-MSCs源性外泌体对MSCs成骨分化的影响以及对HUVECs迁移和成血管的影响；同时比较了MSCs在3D培养（3D-exos）和常规2D培养（2D-exos）下、缺氧和常氧状态下产生的外泌体，在HUVECs血管生成中的生物活性的影响以及HMGB-1在其中的潜在机制。我们研究结果发现：过表达HMGB-1的MSCs成骨能力增强，其过表达HMGB-1的MSCs源性外泌体的与ECs共培养时可以增加ECs的迁移和成血管能力；一系列的体外和体内实验表明，与2D-exos相比，3D-exos对HUVEC细胞增殖、迁移、管腔形成和体内血管生成具有更强的影响。此外，3D-exos的优势可能归因于HMGB-1/AKT信号的激活。同时，缺氧条件下培养的MSCs释放的外泌体促进HUVEC的细胞增殖、迁移和血管生成。缺氧诱导MSCs外泌体中高迁移率族蛋白B1（HMGB-1）的表达增加，沉默HMGB-1则消除了其对HUVEC的血管生成作用。此外，外泌体HMGB-1激活JNK信号通路并诱导缺氧诱导因子-1α（HIF-1α）/血管内皮生长因子（VEGF）表达，从而增强HUVEC中的血管生成。我们的数据显示，外泌体HMGB-1通过JNK/ HIF-1α信号传导促进血管生成。这些结果表明，模拟移植体内血管生成的微环境，三维培养方式、缺氧环境所营造的不同微环境的改变可以促进MSCs源性外泌体产生促血管因子，促HUVEC的血管生成，而且HMGB-1在其中起重要的促进作用。可见，三维培养、缺氧条件下MSCs源性外泌体，联合活性因子HMGB-1具有开发血管生成相关疾病新治疗策略的潜力。