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目的:探索人脐静脉血内皮祖细胞的分离和扩增条件,并观测其生物学特性。方法:采集人脐静脉血,应用密度梯度离心法,分离其中单个核细胞,流式细胞术检测CD133+CD34+阳性率;利用差速贴壁法(48h内贴壁和48h后贴壁)联合内皮细胞专用培养基EGM-2培养细胞,接种于预先包埋了明胶培养瓶或培养板,倒置显微镜观察细胞生长形态和形成集落能力,免疫细胞化学法检测其免疫表型,摄取Ac-LDL和连接UEA-1功能,在生长因子培养体系中诱导其向成熟内皮细胞分化。结果:所获单个核细胞中CD133+CD34+百分比为1.06%;在EGM-2培养体系下可获得2种亚型的内皮祖细胞,即早期内皮祖细胞和晚期增殖性内皮祖细胞。其中48h后贴壁细胞属于早期内皮祖细胞,增殖能力较弱,免疫荧光检测,显示CD14和CD34KDR胞浆呈阳性表达,Ac-LDL+UEA-1+功能特征;而48h内贴壁细胞在10～17d时可见由单个细胞增殖形成的克隆,呈铺路石样单层排列,增殖力旺盛形成融合状态,形成次集集落;经免疫荧光检测,显示CD133CD34和CD34KDR细胞质呈阳性表达,Ac-LDL+UEA-1+功能特征,传代后vWF,CD31呈强阳性表达,是晚期增殖性内皮祖细胞。结论:经人脐静脉血可分离培养获得2种亚型的内皮祖细胞,在特定的培养体系中细胞可由祖细胞表型向成熟内皮细胞分化。
Objective: To explore the conditions of isolation and amplification of human umbilical vein endothelial progenitor cells and to observe their biological characteristics. Methods: Human umbilical vein blood was collected and mononuclear cells were isolated by density gradient centrifugation. The positive rate of CD133 + CD34 + was detected by flow cytometry. The differential adhesion method (48h adhesion and 48h adhesion) Cells were cultured in EGM-2 cell culture medium and inoculated into gelatin culture flasks or culture plates. The growth morphology and colony formation ability of EGM-2 cells were observed by inverted microscope. Immunocytochemistry was used to detect the immunophenotype, UEA-1 function induces its differentiation into mature endothelial cells in a growth factor culture system. Results: The percentages of CD133 + CD34 + in mononuclear cells were 1.06%. Two subtypes of endothelial progenitor cells, early endothelial progenitor cells and late proliferative endothelial progenitor cells, were obtained under EGM-2 culture system. After 48h, adherent cells belonged to early endothelial progenitor cells with weak proliferative ability. Immunofluorescence showed that CD14 and CD34KDR were positive in cytoplasm and Ac-LDL + UEA-1 + ~ 17d, the colonies formed by single cell proliferation were arranged in a monolayer of paving stones. The proliferative force was exuberant to form a fusion state, forming colonies. The expression of CD133CD34 and CD34KDR cytoplasm was positive by immunofluorescence assay. The expression of Ac-LDL + The features of UEA-1 +, vWF and CD31 were strongly positive after passage, which is a late proliferative endothelial progenitor cell. CONCLUSION: Two types of endothelial progenitor cells can be isolated and cultured in human umbilical vein blood. In certain culture system, the cells differentiate into mature endothelial cells by progenitor phenotype.