两阶段法体外诱导人动员外周血CD34^+细胞分化为巨核细胞

A Two-phase Culture System for Megakaryocyte Differentiation of Human Mobilized Peripheral Blood CD34^+ Cells

本研究提出两阶段法巨核细胞分化模型:首先将人外周血来源CD34+细胞在Cocktail或CC100两种增殖培养液中培养3、4、5或6 d,然后转入含有TPO和SCF的分化培养液中继续培养7、8或9 d。培养结束后通过比较细胞增殖、分化及成熟情况,优化培养条件。结果显示最优的诱导分化条件是,CD34+细胞在Cocktail培养液中扩增3 d后转入分化培养液中继续培养7 d;所获得的CD41+细胞和多倍体细胞数量是起始CD34+细胞的16倍和3倍;该条件下获得的CD41+和多倍体细胞数量显著高于常用的TPO法和TPO+SCF培养法。因此,运用本研究优化的两阶段培养模式能获得比单阶段直接诱导法更多的CD41+细胞和多倍体细胞,为巨核细胞和血小板相关的理论、临床研究提供新的更高效的巨核细胞体外扩增、分化模型。

In our study,a two-phase culture system was developed to acquire large amount of CD41＋ and polyploidy cells.Human mobilized peripheral blood CD34＋（PB CD34＋） cells were first cultured in expansion medium（Cocktail or CC100 medium） for 3,4,5 or 6 days,and then cultured in megakaryocytic differentiation medium containing TPO and SCF for additional 7,8 or 9 days.Cell expansion,morphology,CD41＋ cell percentage and DNA content were investigated to evaluate the protocol.The result showed that more CD41＋ and polyploidy cells could be obtained following the two-phase culture with Cocktail medium than with CC100.Moreover,with 3 days expansion in Cocktail medium plus 7 days in differentiation medium,the initial CD 34＋ cells obtained 16-fold expansion of CD41＋ cells and 3-fold expansion of polyploidy cells,such obtained level being significantly higher than that of culturing cells with only one step in TPO or TPO＋SCF.We conclude that with the two-phase culture system,PB CD34＋ cells can expand and differentiate to more CD41＋ and polyploidy cells than those cultured only in accordance to the one-stage culture protocol,so a new and highly efficient megakaryocyte differentiation model for megakaryocyte and platelet related researches is provided already.