2型糖尿病内皮祖细胞来源Exosomes对自身细胞增殖、凋亡及体外成管作用的影响及黄芪组分干预研究

The function of endothelial progenitor cells (EPCs) decline play a key role in the development of diabetic angiopathies.As the transfer of cell information, Exosomes can promote the angiogenesis and proliferation of various cells,But the effect of Exosomes derived from EPCs on the biological behavior of EPCs, has not yet been elucidated. Exosomes derived from EPCs were isolated and purified by serial centrifugation and sucrose gradient centrifugation.characterized exosomes by transmission electron microscopy and western blotting, incubated exosomes with EPCs at different time.The effects of the exosomes on the proliferation and apoptosis f EPCs were analyzed with EdU assay kit and flow cytometry,EPCs migration, adhesive capacity and in vitro vasculogenesis activity were assayed by live cell imaging, adhesion assay and in vitro vasculogenesis kit,respectively.the protein expression of PI3K/Akt/eNOS were detected with Western blotting. After Astragalus Membranaceus intervention,The calcium concentration changes in EPCs were determined by the fluorescence Probe Fura-2 and inverted fluorescence microscope detection system for calcium. The expression level of syntaxin was detected by Western blotting.The purpose of this study is to clarify the effects of exosomes derived from EPCs on the biological behavior of EPCs and explore the mechanism.And to clarify the influence of Astragalus Membranaceus on exosomal excretion of EPCs, Provide some enlightenments for the treatment of diabetic vascular complications.

内皮祖细胞(EPCs)功能下降在糖尿病血管病变的发生发展中起关键作用；Exosomes作为细胞间信息的传递体，可以促进多种细胞的增殖和血管再生，但EPCs来源的Exosomes对EPCs自身生物学行为的影响目前尚未阐明。本课题采用离心超滤和蔗糖密度梯度离心法提取EPCs来源的Exosomes并与EPCs培养不同时间，采用EdU增殖检测法、流式细胞仪检测EPCs的增殖及凋亡；live cell imaging、黏附能力实验和体外血管生成试剂盒检测EPCs的迁移、黏附和体外成管能力；Western Blot检测EPCsPI3K/Akt/eNOS表达。黄芪组分对EPCs进行干预后，检测EPCs胞内Ca2+的变化及膜融合机制相关蛋白表达。旨在阐明EPCs来源的Exosomes对EPCs自身生物学行为的影响及机制，以及黄芪组分影响Exosomes的外泌过程，为糖尿病血管并发症的治疗提供新的治疗思路。

EPCs功能紊乱在糖尿病血管损伤起关键作用，促进EPCs增殖、迁移及血管生成能力的改善，可以增强血管再生和损伤血管的再内皮化过程，对糖尿病患者损伤血管的修复和缺血组织侧枝循环的形成发挥重要作用；Exosomes是细胞与细胞之间交流对话的媒介并作为细胞间信息的传递体，在多种病理生理条件下扮演了十分重要的角色，可以促进多种细胞的增殖和血管再生，但 EPCs 来源的 Exosomes对EPCs自身生物学行为的影响及机制，目前尚未见相关报道。本研究阐明了EPCs来源的Exosomes对EPCs自身生物学行为的影响，明确了EPCs来源的Exosomes对EPCs增殖、迁移、凋亡和血管生成。证明了黄芪组分干预能够影响EPCs来源Exosomes的外泌过程及生物学效应，阐明了黄芪组分对EPCs来源Exosomes总蛋白影响、EPCs细胞胞内Ca 2+的变化及膜融合机制-SNARE相关蛋白syntaxin的表达；阐明了黄芪组分通过影响EPCs来源Exosomes的外泌过程，进一步影响EPCs的调控血管生成和促进损伤内皮修复功能的作用机制，实验成果能为今后应用黄芪治疗糖尿病血管并发症提供理论依据。