脐血来源的两种内皮祖细胞的分离、培养和鉴定  

Isolation,Culture and Identification of Two Types of Endothelial Progenitor Cells from Human Umbilical Cord Blood

作　　者：段华新[1] 卢光琇[2] 程腊梅[2] 

机构地区：[1]广州医学院附属广州市第一人民医院血液科,广东广州510180 [2]中南大学生殖与干细胞工程研究所,人类干细胞国家工程研究中心,湖南长沙410008

出　　处：《中国实验血液学杂志》2008年第2期387-391,共5页Journal of Experimental Hematology

基　　金：国家863项目基金(编号2003AA205181);国家937项目基金(编号00CB51010);国家自然科学基金重点项目(编号30030070);中国博士后基金(20070420762);湖南海上自然科学基金(07JJ5034);广州市医药卫生科技项目(2007-YB-003)资助

摘　　要：为了建立从人脐血分离培养两种内皮祖细胞的方法和培养体系,从脐血分离单个核细胞,在含有内皮细胞条件培养液的体系中培养得到两种细胞,利用摄取乙酰化低密度脂蛋白(acetylated low-density lipoprotein,DiI-Ac-LDL)和结合欧洲荆豆凝集素(Ulex European Agglutinin1,UEA-1)进行鉴定,并进一步用免疫细胞化学、RT-PCR、流式细胞术对它们进行内皮细胞相关的表型检测。结果表明,这两种细胞均能摄取DiI-Ac-LDL和结合UEA-1,表达内皮细胞的标志如CD31、CD144和血管假性血友病因子(von Willebrand Factor,vWF),有CD31、酪氨酸激酶受体(kinase tyrosine insert domain receptor,KDR)、CD144和内皮一氧化氮合成酶(endothelial NO synthase,ENOS)的基因转录。但是,它们的增殖能力明显不同,分别称为循环成血管细胞和高增殖潜能内皮祖细胞。流式细胞计数结果显示,这两种细胞在一些表面标志表达方面也存在明显的差异。结论:利用含内皮细胞条件培养液的培养体系,成功地从脐血分离的单个核细胞中培养得到了两种内皮祖细胞。The aim of this study was to establish the method of isolating and culturing endothelial progenitor cells （EPCs） from human umbilical cord blood. Mononuclear cells （MNCs） from human umbilical cord blood were cultured by using culture system supplemented with endothelial cell-conditioned medium. The obtained two types of cells were purified by picking up colonies, identified by uptake of acetylated low-density lipoprotein （Ac-LDL） and binding to lectin [ Ulex European Agglatinin （ UEA-1 ） ], and were analyzed for the expression of markers by flow cytometry. The results showed that there were significant differences between two types of cells in proliferation, so they were referred as circulating angiogenic cells （ CACs ） and high proliferative potential endothelial progenitor cells （ HPP-EPCs ）, respectively. They were in accordance with the standards of EPCs, could uptake DiI-Ac-LDL and binded to UEA-1, and expressed the markers of endothelial cells, such as CD31, CD144 and vWF detected by immunocytochemistry. The transcription of CD31, KDR, CD144 and ENOS in both of them could be detected by RT-PCR, but FACS analysis showed significant differences of surface marker expression between them. In conclusion, two types of EPCs are successfully obtained by culturing MNCs isolated from human umbilical cord blood using endothelial cell-conditioned medium.

关 键 词：内皮祖细胞 脐血 内皮细胞条件培养液 细胞培养 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]