大鼠外周血单个核细胞体外向内皮细胞诱导分化时的表型变化

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。

Phenotype alterations during the differentiation of rat peripheral blood mononuclear cells into endothelial cells

BACKGROUND: Changes of both stem cell markers and endothelial cell phenotype help understand characteristics of endothelial progenitor cells during adherent differentiation. However, there is still no specific cell marker to distinguish from mature endothelial cells. OBJECTIVE: To study the changes of stem cell markers and endothelial cell phenotype during the differentiation of rat peripheral blood mononuclear cells into endothelial cells. DESIGN, TIME AND SETTING: Cell observation study was performed in the Laboratory of Cardiac Surgery, Qingdao Municipal Hospital between June 2004 and December 2008. MATERIALS: Peripheral blood was drawn from male SD rats to obtain mononuclear cells by Ficoll density gradient centrifugation. METHODS: Mononuclear cells were in vitro cultured in fibronectin culture medium and induced by vascular endothelial growth factors (VEGF) and basic fibroblast growth factors (bFGF) in order to stimulate a differentiation into endothelial cells. MAIN OUTCOME MEASURES: Adherent cells in the culture system were identified for CD31, CD34, Flk-1 and vWF with immunochemistry within 1-7 days. RESULTS: The expressions of CD31, CD34, Flk-1, vWF on adherent cells were different in different time durations. The expressions of CD31 and CD34 started on the 2nd day of culture, reached the peak on the 4th day, gradually decreased and even disappeared on the 6th day. While, Flk-1 expressed on the 3rd day of culture, gradually increased, and reached at the peak on the 7th day. vWF expressed gradually until 100% on the 7th day. CONCLUSION: The differentiation of peripheral blood stem cells into endothelial progenitor cells is characterized by the appearance of endothelial cell phenotypes and the disappearance of stem cell markers, both in the manner of gradual progression.