卵泡刺激素诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

Differentiation of Chicken Embryonic Stem Cells to Male Germ Cells by Follicle-stimulating Hormone

文章探讨了卵泡刺激素(FSH)诱导鸡胚胎干细胞(ESCs)向雄性生殖细胞分化的作用效果。首先对FSH的适宜诱导浓度进行筛选,再将传至2代的ESCs,在RA和支持细胞诱导的基础上添加适宜浓度的FSH进行诱导,通过形态学观察、实时荧光定量PCR及免疫细胞化学对诱导结果进行检测。筛选出适宜的FSH浓度为25 ng/mL。单独使用FSH诱导,未出现类SSCs样细胞;FSH与支持细胞诱导至第6天出现类胚体,至第12天,出现少量精原样细胞;FSH联合支持细胞和维甲酸(RA)进行诱导,至第2天出现类胚体,第12天出现类SSC样细胞。实时荧光定量PCR结果显示,FSH+支持细胞组、FSH+支持细胞+RA组中,Stra8、integrinβ1、Dazl表达量都持续上调,ESCs标记基因Nanog、Sox2表达量均呈持续下调趋势。添加FSH的诱导组中Stra8的表达量相对于各对照组上调显著。FSH单独诱导不能引起ESCs向雄性生殖细胞分化,但具有促进支持细胞和RA诱导ESCs向雄性生殖细胞分化的作用效果,该结果为进一步研究雄性生殖细胞的形成和调控机制提供理论基础。

This study investigated the effect of follicle-stimulating hormone （FSH）on differentiation of embryonic stem cells （ESCs）to male germ cells. Screening the suitable concentration of FSH firstly,then added suitable concentration of FSH into the medium with retinoic acid （RA）and sertoli cells to induce 2 passaged male ESCs. The induced results were acquired by morphology, quantitative polymerase chain reaction （qPCR）and immunocytochemistry. The suitable concentration of FSH was 25 ng/mL. Germ cell-like cells wasn＇t found with only FSH. By inducing ESCs with FSH and sertoli cells,embryoid bodies （EBs） appeared at the sixth day and a few germ cell-like cells could be observed. In the groups that FSH was combined with RA and sertoli cells,EBs appeared at the second day, then EBs broke and germ cell-like ceils could be observed at the twelfth day. The result of qPCR showed in the groups of FSH＋sertoli cells and FSH＋sertoli cells＋RA,the expression levels of germ cell-specific genes Stra8,integrinβ1,Dazl all showed an significant upward trend, while the expression levels of ESCs marker genes Nanog, Sox2 were decreased significantly. Compared with each control group,the expression of Stra8 in the groups with FSH was up-regulated significantly. FSH had an effect on differentiation of ESCs to male germ cells. However,it had an important role in promoting the induced effect of sertoli cells and RA on ESCs differentiate to male germ cells. The results provided theoretical foundation for studying cytogenesis and regulatory mechanism of male germ cells.