摘要
目的:比较从外周血单个核细胞(PBMCs)和CD133免疫磁珠分选体外培养人外周血内皮祖细胞(EPCs)的表型、分化、扩增、功能特点,为EPCs细胞治疗提供临床参考。方法:健康成年人外周血,经Ficoll密度梯度离心法得PBMCs,经直接接种法或免疫磁珠分选CD133阳性细胞后置于M199培养基中培养,于第7、14d比较2组细胞表型变化及细胞因子分泌、扩增、体外血管形成及趋化能力。结果:相同血量标本PBMCs组早期集落数高于CD133+组(P<0.01),随着培养时间的推移,流式细胞检测2组细胞均显示造血干细胞标志的表达下调和内皮细胞标志表达上升,但CD133+组内皮标志CD144表达率低于PBMCs组(P<0.01),ELISA法检测到在PBMCs组早期EPCs分泌VEGF的水平高于CD133+组(P<0.01),MTT法显示PBMCs组有较强的增殖能力,基质胶及Tr-answell实验表明PBMCs组细胞参与血管形成的能力较强。结论:CD133+来源的EPCs分化、分泌、增殖及血管形成能力相对较低,推断PBMCs中CD133-细胞可能在形成功能性的EPCs中发挥更重要的作用。
AIM： To compare the methods of two currently employed isolation methods for endothelial progenitor cells （EPCs） ： from total peripheral blood mononuclear cells （PBMCs） and from enriched CD133^ ＋ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS： PBMCs from the healthy subjects were used for CD133^＋ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS： PBMCs produced more colony -forming units （CFU） than CD133^＋ cells from the same volume of blood （P 〈0.01 ）. From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144^＋ cells in CD133^＋ group were lower than those in PBMCs groups （P 〈 0. 01 ）. Cells in PBMCs group secreted more VEGF than that in CD133^＋ group on 7 d （P 〈0. 01 ）. Compared to CD133^＋ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION： CD133^＋ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that it＇s not a preferential way to obtain EPCs for clinic therapy.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2010年第2期368-373,共6页
Chinese Journal of Pathophysiology
关键词
内皮祖细胞
细胞培养
免疫磁珠分选法
Endothelial progenitor cells
Cell culture
Magnetic activated cell sorting
