人脐带血内皮祖细胞体外培养和鉴定  

Culture and identification of endothelial progenitor cells from human umbilical cord blood in vitro

作　　者：方立建[1] 宋鄂[2] 栾瑛[2] 陆成伟[2] 杨威[2] 石慧[2] 毕明超[2] 

机构地区：[1]北京市房山区良乡医院眼科,北京市102401 [2]吉林大学第一临床医院眼科,吉林省长春市130021

出　　处：《中国组织工程研究与临床康复》2010年第45期8450-8454,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：吉林省博士点基金(2007018311);课题题目:内皮祖细胞眼内移植示踪方法的研究;吉林省科委国际合作(20050705-3);题目:高糖对内皮前体细胞影响的研究~~

摘　　要：背景:传统免疫磁珠法分选内皮祖细胞的方法操作复杂、费用大,细胞获得率较低,且内皮祖细胞的增生及分化受限。目的:尝试改良人脐带血内皮祖细胞体外诱导分化及鉴定的方法,为实现内皮祖细胞移植、改善血管功能提供足量细胞来源。方法:采用密度梯度离心方法获得人带血单个核细胞,体外进行诱导、分化和扩增,通过免疫组织化学、免疫荧光和流式细胞仪等技术对脐血来源的内皮祖细胞进行鉴定。结果与结论:脐带血单个核细胞在培养过程中出现梭形贴壁和铺路石样等形态;在细胞培养至第7天,免疫组织化学显示:CD31、vWF在体外培养过程中的表达阳性;免疫荧光染色表明:(83.0±4.3)%的贴壁细胞双染呈阳性,并且DiI-acLDL标记的内皮祖细胞红色荧光在体外可以持续6周以上;第7天贴壁细胞流式细胞仪分析显示:CD34、CD133和KDR分别为(17.8±3.7)%、(22.1±4.4)%、(81.5±5.0)%。结果提示,实验成功从脐带血单个核细胞中分离培养出内皮祖细胞,在其体外可扩增并向为内皮细胞方向分化。BACKGROUND：The method of traditional immunomagnetic beads sorting endothelial progenitor cells（EPCs） shows complicated operation,with high cost and low cell obtaining rate.Moreover,the proliferation and differentiation of EPCs are limited.OBJECTIVE：To elucidate a method for in vitro induction and identification of EPC from human cord blood monocytes（CBMC）,to realize EPC transplantation,and to provide enough cell source for improving blood vessel function.METHODS：CBMCs were isolated by Percoll density gradient centrifugation from cord blood,in vitro induced to differentiate and amplified,and then identified by immunohistochemical and immunofluorescent staining and flow cytometry.RESULTS AND CONCLUSION：During culture,the cells became spindle-shaped and displayed cobble-stone morphology with outgrowth.On day 7,immunostaining of adherent cells was positive for the cell markers CD31 and vWF.（83.0±4.3）% of attached cells were positive for the double marker DiI-acLDL/FITC-UEA-Ⅰ.The red fluorescence of DiI-acLDL-labeled EPCs lasted for over 6 weeks in vitro.On day 7,flow cytometric analysis showed positive staining of attached cell for CD34,CD133 and KDR（17.8±3.7）%,（22.1±4.4）% and（81.5±5.0）%,respectively.Results indicate that the mononuclear cells from CBMC differentiate into EPCs.It can differentiate into endothelial cells by in vitro amplification.

关 键 词：内皮祖细胞 细胞培养 细胞移植 人脐带血 诱导分化 

分 类 号：R394.2[医药卫生—医学遗传学]