无血清脐血巨核系祖细胞体外扩增的研究

脐血造血干细胞移植后血小板恢复延迟是一大难题,目前认为这主要与脐血中巨核系祖细胞数量不足及脐血巨核细胞分化成熟延迟有关,而将部分脐血进行巨核系祖细胞体外扩增后输注受者体内是有望解决这一难题的重要途径。但适用于临床应用的扩增条件至今仍未确立。本课题采用人脐血单个核细胞(MNC)在无血清培养体系中使用TPO,IL-3,SCF,IL-6等细胞因子进行不同的组合,在培养的0,6,10,14天进行MNC、CD41^ 细胞及CFU-MK数的检测。以寻找最佳的细胞因子组合及最佳的收获时机。结果表明：无血清条件下TPO与IL-3,SCF,IL-6等细胞因子联用可实现脐血巨核系祖细胞有效的体外扩增,各因子组中以TPO IL-3 SCF IL-6组扩增效果为最佳。其CFU-MK数于第10天最多,扩增达6．8倍,CD41^ 细胞扩增达8．8倍。结论：在人脐血MNC无血清培养条件下。TPO IL-3 SCF IL-6组为巨核系祖细胞体外扩增较佳的因子组合。由于TPO IL-3 SCF IL-6组的CFU-MK数于第10天最多,CD41^ 细胞数亦为同期最高,故培养后收获时间宜控制在其体外培养的第10天。

Ex vivo Expansion of Megakaryocyte Progenitor Cells from Human Cord Blood in Serum-free Culture

Prolonged thrombocytopenia is a puzzling problem following umbilical cord blood (CB) transplantation. It might be the result of inadequate megakaryocyte progenitors and the arrest in the megakaryocyte maturation. It is an important method to solve the problem by transfusing ex vivo expanded CB megakaryocyte progenitor cells into the patients to shorten the duration of platelet recovery. However, the most optimal condition of expansion has not been established so far. In the study, cord blood mononuclear cells (MNC) were cultured in serum free medium with TPO, IL 3 , SCF and IL 6. The numbers of MNC, CFU MK and CD41 + cells were measured at 0,6,10 and 14 days, in order to find the best cytokine combination and optimal harvest time point for clinical use. The results showed that the megakaryocyte progenitors most efficiently expanded with the cytokine combination including TPO, IL 3, SCF and IL 6. The time point of maximal CFU MK growth is day 10. At 10 days, the numbers of CFU MK and CD41 + cells expanded by 6.8 and 8.8 fold respectively. In conclusion, in vitro, the cytokine cocktail including TPO, IL 3, SCF and IL 6 was the most optimal cytokine combination which stimulates the ex vivo expansion of megakaryocyte progenitors in CB MNC and serum free medium. The maximal CFU MK colonies were harvested at 10 days, that may be an optimal harvest time for clinical transfusion.