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目的探讨含人脐血来源的间充质干细胞(MSCs)体系在体外对脐血造血干细胞(HSCs)扩增作用。方法(1)用含人脐血MSCs及不同造血生长因子(HGFs)组合的无血清扩增体系对人脐血CD+34细胞进行体外扩增。(2)于扩增前及扩增后第6、12天分别用双色流式细胞仪动态检测HSCs表面抗原标记:CD+34、CD+34CD-38、CD+34CD+3、CD+34CD+19、CD+34CD+33和CD+34CD+41a细胞的含量。(3)按本实验室方法行体外半固体培养,观察扩增前后脐血细胞粒单核细胞集落形成单位(CFUGM)、爆式红系集落形成单位(BFUE)、混合集落形成单位(CFUMix)及高增殖集落形成单位(CFUHPP)集落形成情况。结果(1)含人脐血MSCs体系对脐血CD+34CD38细胞的扩增倍数在第6天和第12天分别为159和437倍。该亚群百分比在单纯因子组扩增第12天时为1.98%,而在含脐血MSCs组为9.98%,明显高于扩增前。(2)集落培养表明,含脐血MSCs组扩增第12天与扩增第6天相比,其CFUMix和CFUHPP的扩增倍数增加,而单纯因子组这两种集落的扩增倍数下降。(3)随扩增天数的增加,两组扩增体系中CD+34CD+3和CD+34CD+41a细胞均明显增加,而CD+34CD+19和CD+34CD+3细胞均明显减少。两组相比,含脐血MSCs组差异更显著。结论(1)含脐血MSCs体系不仅能扩增更原始的造血干/祖细胞(HSPC),且具有在短期内(12d)保持HSCs不耗竭。(2)含脐血MSCs体系对脐血CD+34细胞向定向祖细胞的扩增,主要为髓系及巨核系祖细胞,而对其向淋巴系祖细胞的扩增具有抑制作用。
Objective To investigate the expansion of HSCs derived from human umbilical cord blood-derived mesenchymal stem cells (MSCs) in vitro. Methods (1) Human umbilical cord blood CD + 34 cells were expanded in vitro using serum-free medium containing MSCs and different hematopoietic growth factors (HGFs). (2) The surface antigen markers of HSCs were detected by dual-color flow cytometry before and after 6 and 12 days of amplification respectively: CD + 34, CD + 34CD-38, CD + 34CD + 3, CD + 34CD + 19, CD + 34CD + 33 and CD + 34CD + 41a cells. (3) According to the method of our laboratory, semi-solid culture in vitro was performed to observe the expression of CFU-M, BFUE, CFUMix and High proliferative colony forming units (CFUHPP) colony formation. Results (1) The multiplicity of expansion of cord blood CD + 34CD38 cells in MSCs containing human cord blood was 159 and 437 fold on the 6th and 12th days, respectively. The percentage of subpopulations was 1.98% on the 12th day of simple factor group amplification, and 9.98% on MSCs containing cord blood, which was significantly higher than that before amplification. (2) The colony culture showed that the multiplication of CFUMix and CFUHPP was increased on the 12th day after MSCs were cultured on the 12th day compared with the 6th day on the day of amplification, whereas the fold of multiplication of CFUMix and CFUHPP on the simple factor group was decreased. (3) With the increase of days of amplification, the CD + 34CD + 3 and CD + 34CD + 41a cells in both amplification systems were significantly increased, while the CD34 + CD34 + CD34 + cells were significantly reduced. Compared with the two groups, cord blood MSCs group more significant differences. Conclusions (1) MSCs containing umbilical cord blood can not only expand more primitive hematopoietic stem / progenitor cells (HSPCs), but also keep HSCs not to be depleted in a short period of time (12 days). (2) The proliferation of cord blood CD34 + cells to committed progenitor cells by cord blood MSCs system is mainly myeloid and megakaryocyte progenitor cells, and it has inhibitory effect on the expansion of lymphatic progenitor cells.