BMP4诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

Study on Differentiation of Chicken Embryonic Stem Cells to Male Germ Cells by BMP4

旨在探讨BMP4诱导鸡胚胎干细胞向雄性生殖细胞分化的作用机理。采用单层贴壁培养作为分化模式，分别采用10、20、30和40ng·mL-1 BMP4浓度诱导雄性鸡胚胎干细胞，通过形态学、荧光定量PCR和免疫细胞化学检测诱导效果。根据细胞形态变化和差异基因表达量变化，确定了BMP4的适宜诱导浓度为40ng·mL-1。在适宜浓度诱导过程中，诱导4d类胚体开始出现，6～8d类胚体逐渐增大，数量增多，10d部分类胚体开始散开，变为小的圆形细胞，12d在散开后的类胚体周围可观察到少量生殖样细胞，14d生殖样细胞数目增多，且呈聚团生长的趋势。诱导过程中相应基因表达量也发生变化：胚胎干细胞标记基因Nanog、Sox2表达量显著下降（P〈O．01），生殖细胞特异基因Stra8、Dazl、integrina6、c-kit表达量均呈显著上升趋势（P〈O．01）。BMP4可以引起生殖细胞相应基因的表达，促进雄性鸡胚胎干细胞向雄性生殖细胞方向分化，结果为雄性生殖细胞的体外诱导研究提供试验参考，为进一步研究雄性生殖细胞发生和调控机制提供理论基础。

This study investigated the effect of BMP4 on differentiation of embryonic stem cells （ESCs） to male germ cells. Adherent monolayer culture system was used as a differentiation mode, maleness ESCs were cultured in concentrations of 10,20,30,40 ng mL-1 BMP4 to differ- entiate toward a germ cell lineage. The induced effects were detected by morphology, quantitative polymerase chain reaction（qPCR） and immunocytochemistry. According to the changes in cell morphology and the expression of differential genes, 40 ng mL-1 was identified as the suitable concentration of BMP4 on differentiation. During the induction with 40 ng mL-1 BMP4, embry- old bodies（EBs） began to appear in 4 d and gradually increased in both size and number in 6-8 d, subsequently some of EBs began to disintegrate into small round cells in 10 d, then a small num- ber of germ cell-like cells could be observed in 12 d, and the number increased in 14 d. The ex- pression of corresponding genes also changed during the induction： the expression level of the ESCs marker genes Nanog, Sox2 decreased significantly （P〈0.01）, while the expression levels of germ cell-specific genes StraS, Dazl, integrina6, c-kit all showed a significant upward trend （P〈 0.01）. BMP4 can promote the expression of the corresponding genes of the germ cells, and has an effect on differentiation of ESCs to male germ cells. The result provides references for differ- entiation on male germ cells in vitro and theoretical foundation for studying cytogenesis and regu- latory mechanism of male germ cells.