体外人脐静脉内皮细胞的培养鉴定及抗原表达分析

摘要

目的 探讨体外分离、培养人脐静脉内皮细胞(HUVECs)的方法及进行HUVECs的扩增和鉴定,明确HUVECs表面抗原表达情况.方法 采集正常人脐带,应用改良的酶消化法进行HUVECs的分离培养；通过细胞形态学观察及免疫荧光化学鉴定HUVECs,应用细胞荧光化学进行内皮细胞(ECs)功能检测；应用流式细胞仪分析内皮细胞的特异性抗原表达.结果 观察细胞形态呈铺路石样,Ⅷ因子相关抗原荧光染色阳性,显示出摄取乙酰化低密度脂蛋白、结合荆豆凝集素等ECs的功能特性；流式细胞仪检测CD31、VWF、KDR、CD34抗原表达阳性百分比高,而CD133处于低水平.结论 通过本实验改良的分离培养方法,可以在体外获得较高数量的HUVECs,在细胞形态、表面抗原标志、细胞功能等方面具有ECs特征,可以用于血管ECs模型的构建.%Objective To study the isolation, culture and identification of human umbilical vein endothelial cells (HUVECs) in vitro, and analyze antigens expression. Methods Cultured cells were isolated from umbilical cord by enzyme digestion and were cultured in vitro. Cultured cells were subjected to flow cytometer to analyze the expression of CD31, CD34, VWF, KDR and CD133. Immunofluorescence was performed to detect VWF and dual binding of ac-LDL and UEA-1. Results Cultured HUVECs were like slabstones, which were positively stained for CD31, CD34, VWF, KDR, but negative for CD133. Cultured HUVECs could endocytose ac-LDL and UEA-1. Conclusions HUVECs with the characters of endothelial cells can be cultured abundantly by this method. Purity of HUVECs can be assayed by analyzing antigens expression, which can be used to establish the model of endothelial cells.