脐血造血干细胞向巨核细胞诱导分化的研究

The study on the differentiation and expansion of megakaryocytes from umbilical cord blood derived CD34^+ cells

目的:建立脐血CD34+造血干细胞向巨核细胞诱导分化的体系,探讨最佳的扩增方法。方法:免疫磁珠法分离获得CD34+细胞培养在无血清无基质培养液中,采用TPO加SCF加IL-3加IL-6、TPO加SCF加IL-3、TPO加SCF3种不同因子组合对其诱导分化及扩增。收集3、7、10、14d的扩增产物,运用荧光显微镜检测巨核细胞的表面标志;流式细胞术(FCM)检测巨核细胞的凋亡;并对巨核细胞形成单位(CFU-MK)及DNA含量进行检测。结果:分离获得的CD34+细胞在体外可以有效扩增,随培养时间的延长CD34+/CD41+细胞数第7天达最高值,之后逐渐下降;而CD41+、CD42b+、CD61+细胞随培养时间的延长表达量逐渐增高。加入IL-3和IL-6后,Annexin Ⅴ阳性细胞由(8.26±2.49)%降至(3.51±1.24)%。CFU-MK的数量在第10天时最高,且8倍体及8倍体以上的巨核细胞所占的的百分比增加,即成熟产板型巨核细胞增加。结论:脐血CD34+造血干细胞在体外可向巨核细胞诱导分化及有效扩增。3种因子组合中TPO加SCF加IL-3加IL-6组扩增效率最高。

Objective：To establish the system of the differentiation and expansion of magakaryocytes from CD34＋ cells of unbilical cord blood,and to investigate the best method of expansion.Method：The CD34＋ cells were obtained by immunomagnetic beads methods and cultured in serum-free and stroma-free medium containing the following three different cytokine combinations： thrombopoietin （TPO） ＋stem cellfactor （SCF） ＋interleukin （IL）-3＋IL-6,TPO＋SCF＋IL-3 and TPO＋SCF. We collect the cells at 3rd,7th,10th and 14th day and examine the expression of cell surface molecules by fluorescence microscope. Flow cytometer （FCM） was performed to examine cells apoptosis. We also carried the colony forming unit-megakaryocyte （CFU-MK） assay and maturation evaluation of Mk ploidy.Result：In vitro the CD34＋ cells can expand effectively. With the time of culture the expression of CD41＋,CD42b＋,CD61＋ cells did not change significantly,but the optimal expansion of MK progenitors （CD34＋/CD41＋） was observed at 7th day. The degree of maturation of Mk cells also increased by evaluating the DNA content and CFU-MK assay.Conclusion：In vitro CD34＋ cells of unbilical cord blood can differentiate into magakaryocyte and expand effectively. We belived that the cytokine combination TPO＋SCF＋IL-3＋IL-6 was the optimal expansion method.