人脐血巨核系祖细胞体外扩增的实验研究

Study on the Expansion of Megakaryocyte Progenitors in Vitro from Cord Blood

本研究旨在探讨各种细胞因子对人脐血CD34+细胞体外扩增生成巨核细胞的作用,以建立人脐血巨核系祖细胞体外扩增的最佳体系。采用Ficoll分离液分离脐血单个核细胞,免疫磁珠法分离纯化CD34+细胞,进行体外半固体集落培养和液体培养,观察各种细胞因子组合对CD41+细胞和巨核细胞集落形成单位(CFU-MK)的影响。结果显示:TPO+IL-6+IL3+FLT-3L4因子组合体外培养14天效果最好,在第7、14天CD41+细胞分别扩增了154.67±32.21倍、193.23±25.24倍。结论:本实验建立的巨核系祖细胞体外扩增体系,为促进脐血移植后血小板恢复奠定了实验基础。

This study was aimed to investigate the effect of various cytokines on megakeryocytes expansion in vitrofrom human cord blood CD34^＋ cells in order to establish an optimal culture system for MK expansion. Mononuclear cells were obtained by Ficoll-Hapaque density gradient separation. CD34^＋ cells were positively isolated using a CD34 progenitor cell isolation kit. CD34^＋ cells were placed into 24 well plates at a concentration of 2 × 10^4 per well. Each well contained 1 ml of IMDM with the present of effective MK cells growth cytokines. Clonogenic potentials of MK progenitor were assayed using a methylcellulose cultures system. The results suggested that four cytokines （ IL-3 ＋ IL-6 ＋ TPO ＋FLT3L）culture system could effectively induce and expand cord blood CD41^＋ MK cells. The number of CD41^＋ cellsexpanded 154.67±32.21-fold on day 7, and 193.23±25.24-fold on day 14. In conclusion, established expansion system in vitro for MK cells provides experimental foundation for recovery of platelets after cord blood transplantation.