原代人脐静脉内皮细胞分离和培养方法的改进  

A modified method for the isolation and cultivation of primary human umbilical vein endothelial cell

作　　者：莫薇[1] 王颖智[2] 刘鷖雯[2] 何怡青[2] 杨翠霞[2] 陈晓湘[1] 高锋[2] 

机构地区：[1]广州医学院病原生物学与免疫学教研室,广东广州510182 [2]上海交通大学附属第六人民医院中心实验室,上海200233

出　　处：《热带医学杂志》2011年第7期737-739,742,共4页Journal of Tropical Medicine

基　　金：广东省医学科研基金(B2008068);2009年度广州医学院博士;留学回国人员基金项目

摘　　要：目的建立一套高效、方便、可靠的脐静脉内皮细胞的体外分离和培养方法。方法取剖腹产新生婴儿脐带,加入Ⅰ型胶原酶消化分离人脐静脉内皮细胞,内皮专用培养基EGM-2培养传代。应用相差显微镜观察细胞形态。DiL-Ac-LDL染色证实细胞具有内皮功能。免疫荧光染色证明新分离细胞表达FⅧ相关抗原。流式细胞仪检测细胞表面表达CD31。结果原代培养细胞培养10～15d左右长成单层,细胞呈多角形,铺路石样排列。DiL-Ac-LDL染色阳性,证实细胞具内皮吞噬功能。免疫荧光检测证明细胞特异性表达FⅧ相关抗原。流式细胞仪检测证明细胞表面高表达CD31。结论通过酶灌注法消化脐静脉血管获得细胞,操作简便、高效,EGM-2培养基可保证内皮细胞具有较高纯度,细胞形态学观察和细胞生物学鉴定证明获得人脐静脉血管内皮细胞。Objective To improve the in vitro method for the isolation and identification of human umbilical vein endothelial cells（HUVEC）.Methods 1.Endothelial cells were isolated from freshly obtained human umbilical vein using type I collagenase.The cells were cultured in the EGM-2 medium.2.Morophology of the cells was determined under the phase contrast microscope.3.Endothelial cell function was confirmed by the DiL-Ac-LDL uptaking method.4.Cell surface antigen FVIII was determined using an indirect immunofluorescence staining method and the CD31 expression was analyzed by flow cytometry.Results At 10～15 days after isolateion the cells formed a confluent monolayers and the cells were in polygonal shape.The cells were confirmed as endothelial origin as the cells were found to uptake DiL-Ac-LDL,and expression of FVIII antigen and CD31.Conclusion The developed enzymatic digestion method is simple and effective.To obtain pure endothelial cells,the use of EGM-2 medium is crucial.

关 键 词：脐静脉内皮细胞 细胞培养 鉴定 

分 类 号：Q813.11[生物学—生物工程]