一种从脐血培养高增殖潜能内皮祖细胞的新方法

发布时间：2018-12-06 08:33

【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。
[Abstract]:[objective] to establish a stable and economical method for the culture of umbilical cord blood endothelial progenitor cells (High proliferative potential-endothelial progenitorcells,HPP-EPCs) with high proliferative potential, and to establish a stable and economical method for the proliferation of endothelial progenitor cells (Endothelial progenitor cells,) derived from HPP-EPCs. [methods] umbilical cord blood mononuclear cells were isolated and inoculated into vessels pretreated with fibronectin (Fibronectin,FN). EPC clones derived from HPP-EPCs could be obtained by using MCDB131 medium containing vascular endothelial growth factor (VEGF) and endothelial cell growth additive (ECGS). After 4 days of culture, unadherent cells were removed. After 1021 days of culture, EPC clones derived from HPP-EPCs could be obtained. HUVECs, isolated from human umbilical cord was amplified by MCDB131 medium containing EGF, and human fibroblast (Humanembryonic fibroblast cells,hEFs was isolated and cultured at the same time. The isolated umbilical cord blood EPCs was identified by immunophenotype, UEA1 binding test, Matrigel test and DiI-Ac-LDL phagocytosis test. HUVECs was used as positive control. [results] one HPP-EPC could amplify 108G 1010 EPCs cells in 2 months and maintain normal karyotype in long-term culture in vitro. Both EPCs and HUVECs can express CD31,CD144,vWF, combined with UEA1, and form capillary structure on Matrigel, and can also phagocystify DiI-Ac-LDL. [conclusion] the new method developed in this study can efficiently and economically obtain primitive EPCs, from cord blood. It is expected to be an effective seed cell for cell replacement therapy of ischemic disease.
【作者单位】：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;
【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)
【分类号】：R329

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