一种从脐血培养高增殖潜能内皮祖细胞的新方法  

A new method to cultivate cord blood based high proliferative potential-endothelial progenitor cells

作　　者：孙璇[1,2] 梅花[2] 卢光琇[1] 程腊梅[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,湖南长沙410078 [2]人类干细胞国家工程研究中心,湖南长沙410078

出　　处：《西北农林科技大学学报（自然科学版）》2013年第4期26-32,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家高技术研究发展计划("863"计划)项目(2011AA020113);湖南省科技计划项目(2011FJ3159);长沙市科技计划项目(K1104069-31)

摘　　要：【目的】建立一种培养脐血高增殖潜能内皮祖细胞(High proliferative potential-endothelial progenitorcells,HPP-EPCs)的稳定经济的新方法,并将由HPP-EPCs增殖而来的内皮祖细胞(Endothelial progenitor cells,EPCs)与脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)进行体外比较。【方法】分离脐血单个核细胞,接种到纤连蛋白(Fibronectin,FN)预处理的器皿中,用含有血管内皮生长因子(VEGF)和内皮细胞生长添加物(ECGS)等的MCDB131培养基培养,4d后去除未贴壁细胞,继续培养10～21d后,可以得到HPP-EPCs来源的EPC克隆;从人脐带中分离HUVECs,用含有EGF的MCDB131培养基扩增培养;同时分离培养人成纤维细胞(Humanembryonic fibroblast cells,hEFs)。通过免疫表型、UEA1结合试验、Matrigel试验、DiI-Ac-LDL吞噬试验等对分离的脐血EPCs进行鉴定,以HUVECs为阳性对照,hEFs为阴性对照。【结果】1个HPP-EPC可在2个月中扩增出108～1010个EPCs细胞,在长期体外培养中可以保持正常核型。脐血EPCs和HUVECs均可表达CD31、CD144、vWF,结合UEA1,并能在Matrigel上形成毛细血管样结构,也能吞噬DiI-Ac-LDL。【结论】本研究所建立的新方法能从脐血中高效经济地获得较原始的EPCs,并能在体外长期扩增培养,有望成为缺血性疾病细胞替代治疗有效的种子细胞。[Objective] In this study,we aimed at establishing a stable and economic method to culture cord blood based high proliferative potential-endothelial progenitor cells （HPP-EPCs）, and comparing HPP-EPCs derived EPCs with human umbilical vein endothelial cells （HUVECs） in vitro. [Method] Mononuclear cells （MNCs） separated from human umbilical cord blood were suspended in MCDB131 con- taining VEGF and ECGS before being seeded onto cell culture plate pre-coated with fibronectin. Non-ad- herent cells were removed after 4 days of culture. 10-21 days later, HPP-EPCs forming colonies appeared. H UVECs were separated from human umbilical cord vein,and expanded in MCDB131 containing EGF. We identified the endothelial cell population by sis assay and DiI-acetylated-low density lip immunophenotyping, UEA1 binding assay, Matrigel angiogeneoprotein （DiI-Ac-LDL） uptaken assay. HUVECs were positive control,while hEFs were negative control. [Result] A single HPP-EPC could generate as many as 10^8- 10^10 progenies in vitro within 2 months. They could remain normal karyotype a{ter iong-term culture, They expressed CD31, CD144, vWF, and binding UEA1, and could form capillary-like formation on Matrigel. They also had the ability to incorporate DiI-Ac-LDL. [Conclusion] Our approach is more convenient and economic in culturing and expanding EPCs from umbilical cord blood,which makes EPCs an efficient seed cell type in treatment of ischemia diseases.

关 键 词：高增殖潜能内皮祖细胞 脐血内皮祖细胞 脐静脉内皮细胞 细胞培养 

分 类 号：Q813.1[生物学—生物工程]