成人骨髓源内皮祖细胞的体外分离培养及鉴定

Isolation,culture and identification of adult human bone marrow-derived endothelial progenitor cells in vitro

目的探讨成人骨髓源内皮祖细胞(EPCs)体外分离培养的可行性。方法抽取成年男性骨髓,体外全骨髓培养,传代后采用免疫微珠分选方法收集CD1+33细胞,EGM-MV2条件培养基诱导培养EPCs,观察细胞形态、生长情况;采用流式细胞技术检测分选细胞纯度,免疫荧光法检测EPCs特殊分子标志物CD34、CD133和VE-cadherin表达,透射电子显微镜观察分选细胞超微结构,UEA-1和Dil-ac-LDL双染色法检测分选细胞的吞噬功能。结果分选后细胞培养第3天出现集落样生长,集落边缘细胞形态伸展呈梭形或多边形,传代后呈现串珠样排列;培养至5～6 d,细胞连接成大片条索状结构;CD 3+4、CD 1+33细胞百分率分别为24.13%、93.29%,其表面特异性表达CD133、CD34、VE-cadherin,具有EPCs形态特征,能吞噬Dil-ac-LDL并结合UEA-1。结论成人骨髓来源的EPCs经体外培养后形态、增殖率、生存能力、表面标志表达、功能等均较为稳定,可作为心血管组织工程种子细胞或用于干细胞治疗。

Objective To investigate the feasibility of adult human bone marrow-derived endothelial progenitor cells （EPCs） isolated and cultured in vitro. Methods Adult human bone marrow was extracted, then the whole marrow was cultured in vitro. After primary cells were passaged, CD133 cells were sorted by microheads instrument and cultured with EGM-MV2. The cell growth curve was drawn after separation and passage to observe cell growth capacity. Flow cytometry was used to detect the purity of CD34^＋ and CD133^＋ cells. Cell specific molecule CD133 , CD34, and VE-cadherin expression were detected by immunofluoreseence. Transmission electron microscope was used to observe the ultrastructure of sorted cells. FITC labeled UEA-1 and Dil labeled ac-LDL were used to label cells and detect cell phagocytosis. Results The human bone marrow CD133^＋ cells began to form cell clusters 3 days after cell sorting and cell morphology formed typical string-of- beads-like 3 days after passage; cells went into platform phase on day 5-6 after passage. The rate of CD34^＋ and CD133^＋ cells were 24.13% and 93.29% respectively, the cells presented CD133, CD34 and VE-cadherin. The cells could phagocytose Dil-ac-LDL and combine with UEA-1. Conclusions EPCs isolated and cultured from human bone marrow showed many stable biological characteristics, such as modality, proliferation rate, cell growth capacity, expression of surface marker and function, and may be used as a kind of ideal cell for tissue engineering or stem cell therapy.