c-myb对胚胎干细胞体外造血分化及定型的影响

Roles of c-myb in haemopoietic differentiation and commitment of embryonic stem cells in vitro

目的 通过体外胚胎干细胞培养体系对ES细胞进行c- myb等位基因打靶,建立c -myb+ +及c -myb ES细 胞模型的基础上,探讨转录因子c- myb在造血定型和分化中的作用。方法 通过体外ES细胞去除LIF半固体介质EBs的 造血分化体系,采用甲基纤维素克隆形成分析和real timePCR观察c- myb+ +及c -myb ES细胞,比较分析其不同分化过 程及阶段中造血各系形成和相关基因表达变化。结果 c -myb+ +及c -myb ES胚胎体(EBs)大小及形态相似,c -myb+ +ESEBs形成显著多于c -myb 。c -myb+ +组CFU- E出现在第6天,高峰为第8天;c- myb 组也有相似的改变,但其 CFU- E数目少于c -myb+ +。c -myb+ +组CFU- M高峰为第7天;c- myb 也有相似的改变,但其CFU- M数目高于c- myb+ +。c -myb+ +CFU -GM出现在第7天,高峰为第12天,而c -myb 未见CFU -GM形成。real timePCR分析显示,c -myb 及 c- myb+ +β globin,zeta globin,Lys,C- fms基因表达无显著变化。结论 低水平的c -myb不影响祖细胞的扩增,但影响其 终末分化。低水平的c -myb表达在造血定型中对红系的发育具有一定的影响,而对巨噬细胞的发育成熟无明显影响。 c -myb的表达水平对造血分化相关基因的表达没有明显的影响。

Objective The c-myb is an important transcription factor in early haemopoietic system development and differentiation. We wish to use ES cell culture system by gene target getting cell models of c-myb+/+ and c-myb-/- ES in vitro in order to examine the detailed roles of the transcription factor c-myb in haemopoietic commitment and differentiation. Methods Haemopoietic differentiation was initiated by seeding ES cells in methylcellulose medium in the absence of LIF. The embryoid bodies of c-myb+/+ and c-myb-/- ES were analyzed by methylcellulose colony assay and real-time PCR to compare the formation of each haemopoietic system on different differentiation stage and procedure and relative gene expression. Results The formation of embryoid bodies was similar for both c-myb+/+ and c-myb-/- ES cells with the exception that the size and frequency of EBs reduced in the case of the c-myb-/- cells. The number of embryoid bodies increased more markedly in c-myb+/+ than that in c-myb-/-. Erythroid progenitors (CFU-E) were first present in c-myb+/+ group at 6 d after in vitro differentiation, and their number reached the peak at 8 d. Similar kinetics were seen for the formation of CFU-Es in c-myb-/- group, but their number was lower than that in c-myb+/+ group, and the colonies were generally small. CFU-M were first detectable at 7 d, and the peak levels were present at 7 d in c-myb+/+ group. Similar kinetics were seen for the formation of CFU-M in c-myb-/- group, but their number was lower than that in the c-myb+/+ group. CFU-GM was first detectable at 7 d, and the peak levels were present at 12 d in c-myb+/+ group. CFU-GM was not detected in c-myb-/- group. Real-time PCR analysis showed that there was no change in the gene expressions of β-globin, zeta-globin, Lys, and C-fms in the two groups of c-myb+/+ and c-myb -/-. Conclusion Low levels of c-myb are suff-icient to allow progenitor expansion, but progression of progeniters towards terminal differentiation is significantly altered. Low levels of c-myb can influence erythropoiesis development, but precursor cells capable of differentiating into macrophages are present in haemopoietic commitment. The levels of c-myb can not change the gene expressions of β-globin, zeta-globin, Lys, and C-fms.