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目的通过基因打靶建立c-myb+/+和c-myb+/-胚胎干细胞(ES)模型,了解转录因子c-myb在造血定型和分化中的作用。方法采用去除白血病抑制因子的甲基纤维素体外ES细胞诱导分化体系进行造血分化,收获c-myb+/+和c-myb+/-(表达55%的c-myb)胚胎体进行甲基纤维素克隆形成分析。运用实时定量PCR比较分析c-myb+/+和c-myb+/-造血分化过程的造血相关基因表达。结果c-myb+/+和c-myb+/-ES细胞胚胎体形成相似,但c-myb+/-组胚胎体体积小于c-myb+/+组。两组红系克隆形成单位(CFU-E)克隆形成相似,但c-myb+/-组CFU-E克隆小于c-myb+/+组。两组红系爆式克隆形成单(BFU-E)均出现于第7天,高峰在第10天。两组巨噬系克隆形成单位(CFU-M)形成相似,但c-myb+/-组CFU-M数目多于c-myb+/+组。c-myb+/-组粒-单系克隆形成单位(CFU-GM)形成数目少于c-myb+/+组。PCR分析显示c-myb+/+和c-myb+/-组β-globin、ζ-globin、GATA3、CD34、Lys、c-fms和c-mpl基因表达没有明显差异。结论亚表达水平的c-myb可以满足祖细胞扩增的需要,但却影响其终末分化。亚表达水平c-myb影响前体细胞向红系和粒系的分化,但不影响巨噬细胞的分化。c-myb表达水平不影响β-globin、ζ-globin、GATA3、CD34、Lys、c-fms和c-mpl基因的表达。
Objective To establish c-myb + / + and c-myb +/- embryonic stem cell (ES) models by gene targeting to understand the role of transcription factor c-myb in hematopoiesis and differentiation. Methods The hematopoietic differentiation of ES cells induced by leukemia inhibitory factor in vitro was induced by ES cells. The c-myb + / + and c-myb +/- embryos (55% c-myb) were harvested for methylcellulose cloning Form an analysis. The hematopoietic related gene expression of c-myb + / + and c-myb +/- hematopoietic differentiation was analyzed by real-time quantitative PCR. Results The embryoid bodies of c-myb + / + and c-myb +/- ES cells were similar, but the volume of embryos in c-myb +/- group was smaller than that of c-myb + / + group. The CFU-E clone formation was similar between the two groups, but the CFU-E clone in the c-myb +/- group was smaller than the c-myb + / + group. Two sets of erythroid blast clone form (BFU-E) appeared on the 7th day with the peak on the 10th day. The formation of macrophage colony-forming units (CFU-M) was similar in both groups, but there were more CFU-M in c-myb +/- group than in c-myb + / + group. The number of c-myb +/- clones-monophasic clonogenic units (CFU-GM) formed less than the c-myb + / + group. PCR analysis showed no significant differences in β-globin, ζ-globin, GATA3, CD34, Lys, c-fms and c-mpl gene expressions in c-myb + / + and c-myb +/- groups. Conclusion The sub-expression level of c-myb can meet the needs of progenitor cell expansion, but it affects its terminal differentiation. Sub-expression levels of c-myb affect the differentiation of precursor cells into erythroid and granulocytic lines, but do not affect macrophage differentiation. The expression of c-myb did not affect the expression of β-globin, ζ-globin, GATA3, CD34, Lys, c-fms and c-mpl genes.