Position: Home > Articles > A new method to cultivate cord blood based high proliferative potential-endothelial progenitor cells
Journal of Northwest A & F University(Natural Science Edition)
2013,41
(4)
26-32
一种从脐血培养高增殖潜能内皮祖细胞的新方法
作 者：
孙璇;梅花;卢光琇;程腊梅
单 位：
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
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
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。
译 名：
A new method to cultivate cord blood based high proliferative potential-endothelial progenitor cells
作 者：
SUN Xuan1,2,MEI Hua2,LU Guang-xiu1,CHENG La-mei1(1 Institute of Reproductive and Stem Cell Engineering,Central South University,Changsha,Hunan 410078,China; 2 National Engineering and Research Center of Human Stem Cell,Changsha,Hunan 410078,China)
关键词：
HPP-EPCs;cord blood EPCs;HUVECs;cell culture
摘 要：
【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 containing VEGF and ECGS before being seeded onto cell culture plate pre-coated with fibronectin.Non-adherent cells were removed after 4 days of culture.10-21 days later,HPP-EPCs forming colonies appeared.HUVECs were separated from human umbilical cord vein,and expanded in MCDB131 containing EGF.We identified the endothelial cell population by immunophenotyping,UEA1 binding assay,Matrigel angiogenesis assay and DiI-acetylated-low density lipoprotein(DiI-Ac-LDL) uptaken assay.HUVECs were positive control,while hEFs were negative control.【Result】 A single HPP-EPC could generate as many as 108-1010 progenies in vitro within 2 months.They could remain normal karyotype after long-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.
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