脐血CD34^＋细胞向巨核细胞的分化扩增及基因的表达变化

Differentiation and expansion of megakaryocytes from CD34^+ cells derived from umbilical cord blood and change of gene expression

目的研究脐血CD34+细胞向巨核细胞的诱导分化及体外扩增,并观察此过程中巨核细胞特异性基因表达的变化。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基中,采用TPO+SCF+IL-3+IL-6、TPO+SCF+IL-3、TPO+SCF三种不同因子组合对其诱导分化及扩增。收集3、7、10、14 d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;对巨核细胞进行DNA含量检测以及荧光定量PCR检测特异性基因表达的变化。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。DNA含量检测发现,随着培养天数的增加,多倍体细胞所占的百分比增加。三种因子组合中TPO+SCF+IL-3+IL-6组扩增效率最高。荧光定量PCR显示转录因子GATA-1、NF-E2、TXS、GPIIb和HPRT在第7天表达量最高,之后下降。凋亡转录因子APAF-1随培养天数的延长表达量增加。结论脐血CD34+细胞在体外可向巨核细胞诱导分化及扩增,其基因表达的变化也支持这一结论。

Objective To investigate the differentiation and expansion of megakaryocytes from CD34＋ cells of umbilical cord blood and observe the change of their specific gene expression.Methods CD34＋ cells were obtained by the immunomagnetic bead method and cultured in serum-free and stroma-free media containing the following three cytokine combinations： thrombopoietin（TPO） ＋stem cell factor（SCF）＋interleukin（IL）-3＋IL-6,TPO＋SCF＋IL-3 and TPO＋SCF.The cells were collected on the 3rd,7th,10th and 14th day and expression of cell surface molecules was determined by fluorescence microscopy,and then cell apoptosis was determined by flow cytometery（FCM）,maturation evaluation of the Mk ploidy was also carried out,and specific gene expression was determined by real time fluorescent quantitation PCR.Results In vitro the CD34＋ cells were effectively expanded.With the increase of time,expressions of CD41＋,CD42b＋,and CD61＋ cells did not significantly change,but the optimal expansion of the Mk progenitors（CD34＋/CD41＋） was observed at day 7.Degree of maturation of the Mk cells also increased by evaluating the DNA content.TPO＋SCF＋IL-3＋IL-6 was the optimal expansion method.Real-time fluorescent quantitation PCR displayed that transcription factor GATA-1,NF-E2,TXS,GPIIb and HPRT reached high levels at the 7th day,butAPAF-1 was directly increased with the increase of time.Conclusion In vitro CD34＋ cells of umbilical cord blood could be differentiated into megakaryocytes and effectively expanded,which was supported by gene expression changes.