睾酮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 Testosterone

作　　者：张晨[1] 蒋一秀 王颖洁[1] 赵瑞丰[1] 王曼[1] 张文慧[1] 靳锴 汪怡临 张亚妮[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院江苏省动物繁育与分子设计重点实验室,江苏扬州225009

出　　处：《中国家禽》2015年第11期12-18,共7页China Poultry

基　　金：国家自然科学基金项目(31272429;31472087;31301959);高等学校博士学科点专项科研基金资助课题(20123250120009);中国博士后基金(2012M511326);江苏高校优势学科建设工程资助项目

摘　　要：试验旨在探讨睾酮对鸡胚胎干细胞(ESCs)向雄性生殖细胞分化过程中的诱导作用效果。首先对睾酮的适宜诱导浓度进行筛选,再联合支持细胞和维甲酸(RA)对传至2代的ESCs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。结果表明:单独使用睾酮诱导,未出现精原干细胞(SSCs)样细胞;睾酮与支持细胞联合诱导时,在第6天有少许类胚体出现,至第12天,有少量SSCs样细胞;睾酮联合支持细胞和RA诱导过程中,诱导第2天出现类胚体,诱导第12天后出现类SSCs样细胞。实时荧光定量PCR结果显示,睾酮+支持细胞组中,ESCs标记基因Nanog、Sox2表达量均呈持续下调趋势,Dazl、integrinβ1、Stra8表达量持续上调;睾酮+支持细胞+RA共同诱导组中,各标记基因与ESCs向生殖细胞分化过程中各基因表达的趋势一致,并且Dazl的表达量明显高于对照组。睾酮单独诱导不能使ESCs向雄性生殖细胞分化,但有促进支持细胞和RA诱导效果的作用,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 was aimed to investigate the induction effects of testosterone on the differentiation process of embryonic stem cells （ESCs）into male germ cells. Firstly,the suitable concentrations of testosterone were assessed,then they were added into the medium with retinoic acid （RA）and Sertoli cells to induce male germ cells from ESCs. The results showed that with testosterone alone,SSCs-like cells didn＇t appear,while combined with sertoli cells,some embroid bodies appeared on the sixth day and SSCs-like cells appeared on the twelfth day. Moreover,in testosterone combined with Sertoli cells and RA group,embroid bodies were found on the second day and SSCs-like cells were found on the twelfth day. The result of quantitative polymerase chain reaction showed that expression levels of the ESCs marker genes Nanog, Sox2 decreased significantly, while the expression levels of germ cell-specific genes Stra8, integrin^l, Dazl all showed an significant upward trend in the group of testosterone and Sertoli cells. In the group of testosterone combined with Sertoli cells and RA, the expression of each marker gene was in accord with theirs in the differentiation process of ESCs to germ cells, and the expression of Dazl in the induced group was higher than that in the control group. Although testosterone couldn＇t help the differentiation of ESCs to male germ ceils alone,it could enhance the induced effects of Sertoli cells and RA. The results provided theoretical foundation for studying cytogenesis and regulatory mechanism of male germ cells.

关 键 词：鸡胚胎干细胞 雄性生殖细胞 分化 睾酮 

分 类 号：S814.6[农业科学—畜牧学]