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背景:内皮祖细胞在贴壁分化过程中其干细胞标记以及内皮细胞表型的变化过程将有助于了解内皮祖细胞的分化特性。但目前还未找到区别于成熟内皮细胞特异性的细胞标记。目的:观察大鼠外周血单个核细胞外分化为内皮细胞的过程中干细胞标志物以及内皮细胞表型的变化。设计、时间及地点:细胞观察实验,于2004-06/2008-12在青岛市市立医院心脏外科实验室完成。材料:抽取雄性SD大鼠外周血,应用Ficoll密度梯度离心的方法获得单个核细胞。方法:单个核细胞体外培养于纤连蛋白处理的培养皿中,同时应用血管内皮生长因子和碱性成纤维细胞生长因子诱导,使之向内皮细胞转化。主要观察指标:以免疫组织化学方法检测细胞CD31、CD34、Flk-1、vWF在第1~7天的表达。结果:贴壁细胞的CD31、CD34、Flk-1、vWF在不同时段呈不同程度的表达。干细胞标志物CD31、CD34在培养的第2天开始表达,第4天最强,以后逐渐减弱,第6天消失。而Flk-1在第3天开始表达,以后逐渐增强,第7天时最强。vWF表达是逐步出现的,第7天时表达为100%。结论:大鼠外周血干细胞向内皮祖细胞分化的过程中,干细胞标志物逐渐消失,内皮细胞的表型逐步出现。
BACKGROUND: The process of endothelial progenitor cells differentiating into stem cells and the phenotype of endothelial cells during adherent differentiation will help to understand the differentiation characteristics of endothelial progenitor cells. However, no specific cell marker specific to mature endothelial cells has been found so far. OBJECTIVE: To observe the changes of stem cell markers and the phenotype of endothelial cells during the differentiation of rat peripheral blood mononuclear cells into endothelial cells. DESIGN, TIME AND SETTING: Cell observation experiments were performed at the Cardiac Surgery Laboratory of Qingdao Municipal Hospital from June 2004 to December 2008. MATERIALS: Peripheral blood of male Sprague-Dawley rats was collected and mononuclear cells were obtained by Ficoll density gradient centrifugation. Methods: Mononuclear cells were cultured in fibronectin-treated Petri dishes and induced by vascular endothelial growth factor and basic fibroblast growth factor to transform them into endothelial cells. MAIN OUTCOME MEASURES: The expressions of CD31, CD34, Flk-1 and vWF were detected by immunohistochemistry on days 1 to 7. Results: The expressions of CD31, CD34, Flk-1 and vWF in adherent cells were different at different time points. The stem cell markers CD31 and CD34 began to express on the second day of culture, the strongest on the fourth day, then gradually weakened and disappeared on the sixth day. However, Flk-1 began to express on the third day and gradually increased thereafter, strongest on the seventh day. vWF expression is progressive and expresses 100% on day 7. Conclusion: During the differentiation of rat peripheral blood stem cells into endothelial progenitor cells, the stem cell markers gradually disappear and the endothelial cell phenotype gradually appears.