【作者】 曹烨；

【导师】 严志强；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2007， 硕士

【摘要】 血管内皮损伤后的修复对于延缓和防止心血管疾病的发生发展具有重要意义。当血管内膜损伤时,循环血中的内皮祖细胞(endothelial progenitor cells,EPCs)迁移到损伤部位时就会与暴露的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)接触,受到VSMCs和血液动力学的影响,进而分化为内皮细胞(endothelial cells,ECs),参与损伤内皮的修复。因此,研究EPCs的分化行为,应该考虑血流力学因素及VSMCs的影响。本文采用密度梯度离心法和差异贴壁法从人脐血中获得EPCs,并以DiL-acLDL和UEA-lectin双色荧光法鉴定。应用EPCs与VSMCs联合培养的平行平板流动腔系统,对EPCs施加5 dynes/cm2切应力,观测切应力和VSMCs对EPCs分化的影响。同时,流式细胞技术检测了细胞表面标志CD133、CD34、CD31和vWF的表达率变化。免疫印迹法定量检测Akt蛋白的表达量变化,并且用Akti抑制Akt磷酸化表达,结合流式细胞技术检测Akti预处理后上述各种细胞表面标志的表达率。细胞粘附实验检测EPCs的粘附并以克隆形成单位计数评价EPCs的增殖。结果发现:①VSMCs使EPCs形态略微伸长并上调了ECs表面标志CD31和vWF的表达率,同时下调了祖细胞表面标志CD133和CD34的表达率;②切应力使EPCs形态显著伸长并提高了CD31和vWF的表达率,同时显著降低了CD133和CD34的表达率;在联合培养加力的条件下,切应力对上述各种表面标志表达率的影响更为明显,其中CD31与联合静止组相比上调了330 %,vWF上调了29 %;③各组p-Akt的表达率变化与ECs表面标志CD31和vWF的表达率变化趋势相同,与联合培养加力组相比,添加Akti的联合加力组的CD31和vWF的表达率明显下降,而CD133和CD34的表达率则显著升高;④与VSMCs联合培养组EPCs的粘附比单独培养时上升了63 %;而克隆形成单位数量下降了约60 %。上述结果表明,VSMCs和流体切应力分别能够促进EPCs向ECs分化,两者协同作用的效果更为显著;同时,在联合培养条件下,Akt的活化参与了流体切应力对EPCs分化的影响。VSMCs促进联合培养的EPCs粘附,抑制其增殖。更多还原

【Abstract】 Background: Endothelial progenitor cells (EPCs) is a population of pluripotent cells, which have a crucial role in maintaining the integrity of the endothelium, but the mechanisms regulating the differentiation of EPCs have not been understood completely. In this study the influence of shear stress and vascular smooth muscle cells (VSMCs) on the differentiation of EPCs, as well as a role of Akt was detected in a coculture model. Moreover, the adhesion and the proliferation of EPCs were examined under coculture condition.Method: Human EPCs were separated from cord blood and identified by double staining for the fluorescent labeled ac-LDL and UEA-lectin. VSMCs/EPCs coculture system were made up by a parallel-plate coculture flow chamber. Flow cytometry assessed the expression of CD133, CD34, CD31 and von Willebrand factor (vWF). Activation of Akt was detected by western blot. The adhesive ability of EPCs was examined by adhesion experiment and the proliferation of EPCs was detected by the number of clone forming units (CFUs).Results: Shear stress and VSMCs increase expression of CD31, vWF, but simultaneously decrease expression of CD133, CD34 in EPCs respectively. The influence of shear stress is more significant when EPCs were cocultured with VSMCs. Akt was activated in EPCs by shear stress or VSMCs or both. Furthermore, when blockage of Akt with its inhibitor Akti in the cocultured EPCs exposed to shear stress both CD133 and CD34 were upregulated, and CD31 and vWF were downregulated significantly. The adhesion of EPCs was upregulated by 63% and the number of CFUs increased by 60%.Conclusions: The results demonstrate that shear stress and VSMCs promote endothelial differentiation of EPCs via activation of Akt. Moreover, the adhesion of EPCs was upregulated but the proliferation of EPCs was downregulted.更多还原