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目的分离脐血干/祖细胞(CD34+CD38-)进行体外长期培养,观察分析其增殖、细胞表面分子标志和染色体核型的特征。方法用流式细胞仪分选CD34FITC和CD38PE标记的CD34+CD38-脐血原始细胞,在含细胞生长因子IL3、IL6、GMCSF、EPO、SCF和胰岛素样生长因子的干细胞培养基中培养6个月,用流式细胞术检测体外培养30d的干/祖细胞表面标记,并用G显带方法分析其染色体核型。结果在一定培养条件下,经7～12d培养,脐血干/祖细胞(CD34+CD38-)开始增殖。培养6个月后,每孔接种1个细胞,细胞数增殖至250～350个;每孔接种10个细胞,细胞数可增殖至400～500个。每孔接种1个细胞其细胞增殖峰持续时间(8～9代)比接种10个细胞(6～7代)长。经体外长期培养增殖,细胞仍强烈显示干/祖细胞表面分子标记(CD34+CD38-);细胞染色体数目、结构未见异常。结论脐血干/祖细胞(CD34+CD38-)经体外特异性培养增殖,可为大量脐血干/祖细胞移植提供细胞来源。
OBJECTIVE: To isolate human umbilical cord blood stem / progenitor cells (CD34 + CD38-) for long-term culture in vitro and observe the characteristics of their proliferation, cell surface molecular markers and karyotypes. Methods CD34FITC and CD38PE-labeled CD34 + CD38-cord blood blast cells were sorted by flow cytometry for 6 months in stem cell medium containing the cytokines IL3, IL6, GMCSF, EPO, SCF and insulin-like growth factor The surface markers of stem / progenitor cells cultured in vitro for 30 days were detected by flow cytometry and their karyotypes were analyzed by G banding method. Results Under certain culture conditions, cord blood stem / progenitor cells (CD34 + CD38-) began to proliferate after being cultured for 7-12 days. After culturing for 6 months, one cell per well was inoculated and the number of cells proliferated to 250-350 cells. After inoculating 10 cells per well, the number of cells could proliferate to 400-500. Each cell inoculation of a cell proliferation peak duration (8 to 9 generations) than inoculated 10 cells (6 to 7 generations) longer. Long-term in vitro proliferation, the cells still strongly show the surface of the stem / progenitor cell markers (CD34 + CD38-); the number of chromosomes, the structure was no exception. Conclusion The cord blood stem / progenitor cells (CD34 + CD38-) proliferate in vitro and provide a source of cells for transplantation of a large number of cord blood stem / progenitor cells.