修饰人脐血来源的内皮祖细胞外泌体的功能促进糖尿病血管损伤后内皮化及其机制研究

Patients with critical limb ischemia are at risk for restenosis and complications arising from infrapopliteal arterial revascularization with drug-eluting device. Endothelial progenitor cells (EPCs) transplantation can reduce restenosis by promoting re-endothelialization， but it is challenged by microvascular thrombus and potential inducing neoplasm. Our previous study indicated that exosomes derived from EPCs accelerated re-endothelialization by enhancing endothelial function. In order to promote diabetic endothelia cell repair, we should solve the problems of less circulating EPCs and the impaired regenerative capacity of endothelial cells. We hypothesized that exosomes with over- expression of both CXCR4 and mircroRNA-126 could accelerate re-endothelialization by enhancing the migration and proliferation of endothelial cells and mobilization of EPCs as well. In this study, lentiviral vectors with over expressing mircroRNA-126 and CXCR4 were transfected to human umbilical cord blood endothelial progenitor cells , and their exosoms with over- expression of both CXCR4 and mircroRNA-126 were extracted,purifed and obtained. Subsequently, the effects of exosomes on vascular endothelial cells(including migration and proliferation) both under normal glucose and high glucose were investigated in vitro after the exosomes treatment. In addition, induction of the endothelial injury was performed in the diabetic rats’ carotid artery, and the pro-re-endothelialization as well as number of EPCs derived from peripheral blood after exosomes treatment were measured. Finally, SDF-1αlevel in the supernatant EPC cultures, blood and vascular injury areas were assessed to find out whether exosoms with over- expression of both CXCR4 and mircroRNA-126 can promote re-endothelialization by establishing an self-amplifying feedback loop of the SDF-1α/CXCR4 axis.

因内皮化延迟，药物洗脱球囊治疗膝下动脉病变面临挑战。干细胞移植虽然可以促进内皮化，但具有栓塞血管、致瘤及致畸风险。我们的前期研究表明，人脐血来源的内皮祖细胞的外泌体可以促进内皮细胞的迁徙、增殖而加速内皮化。要促进糖尿病血管内皮化还须克服内皮祖细胞数目少、内皮细胞MicroRNA-126含量低而增殖功能障碍的缺点。我们设想通过体外构建高表达CXCR4和MicroRNA-126的外泌体，经静脉注射后定向聚集至血管损伤局部，形成调控SDF-1α/CXCR4轴正反馈回路来实现。本研究应用慢病毒转染脐血来源内皮祖细胞形成的高表达CXCR4和MicroRNA-126的外泌体，通过检测内皮细胞的迁徙和增殖、内皮祖细胞的动员、糖尿病大鼠颈动脉损伤后内皮化的程度，以及血管内皮细胞、外周血以及骨髓的SDF-1α的变化，明确修饰后的外泌体可趋向性聚集至血管损伤局部、促进糖尿病大鼠血管损伤后内皮化的价值和机理。

背景：实现糖尿病血管损伤后内皮化必须克服内皮祖细胞数目少、内皮细胞MicroRNA-126含量低而增殖功能障碍的缺点。.主要研究内容和结果: 用携带CXCR4 的慢病毒质粒（lenti-vector）转染HUCB-EPCs，获得CXCR4 修饰的外泌体, 流式细胞仪结果显示CXCR4-EPCs中 CXCR4表达95.8%，对照PLVX-EPCs转CXCR4表达8.7%。过表达CXCR4的内皮细胞外泌体（CXCR4-Exo）较EPC-Exos、PLVX-Exos促进HUVEC细胞增殖、成管效果更显著；划痕实验结果显示， CXCR4-Exos较对照组、EPC-Exos、PLVX-Exos可以显著促进HUVEC迁移。.miRNA-mRNA联合分析显示，miR-21-5p在EPCs外泌体中高度富集；用Miranda和RNAhybrid软件对EPCs外泌体及PBS处理后内皮细胞差异表达中预测其靶基因，结果血小板反应蛋白（Thrombospondin-1， THBS1）是其唯一的靶基因。加入miR-21-5p抑制剂后，THBS1 mRNA和蛋白质水平较EPCs外泌体处理组显著升高。通过GO富集进一步分析表明这些基因参与了与血管发育相关的几个生物学过程。体外功能缺失分析实验验证了这一结果。因此，我们结果表明EPC–exosomes通过miR-21-5p抑制了THBS1 的表达而促进内皮细胞的迁移和增殖。.然而，内皮祖细胞的外泌体并不能很好地在糖尿病小鼠动脉损伤模型中发挥功能，我们探索了糖尿病状态下其它参与血管修复信号通路。我们发现在糖尿病状态下升高的Sema3E以未切割的形式为主，且未切割的形式较切割形式在体外实验中具有更强的抑制人主动脉内皮细胞增殖的能力。此外，Sema3E的受体PlexinD1，在血管损伤部位边缘内皮的PlexinD1表达增高，给予高浓度PlexinD1中和的组别其动脉内膜愈合速度大大高于对照组别。抑制Sema3E/PlexinD1信号通路有助于改善内膜延迟愈合。