视黄酸诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

doi:10.16431/j.cnki.1671-7236.2020.07.019

摘要/Abstract

摘要： 体外胚胎干细胞（embryonic stem cells，ESCs）向生殖细胞分化可用于治疗不育症，同时也为揭示种系世代的分子机制提供最佳模型。试验旨在探讨视黄酸（retinoic acid，RA）诱导鸡胚胎干细胞（chicken embryonic stem cells，cESCs）向雄性生殖细胞（male germ cells，MGCs）分化的作用效果。利用胰蛋白酶消化法从新鲜种蛋X期鸡胚中分离胚胎干细胞，以鸡胚成纤维（chicken embryo fibroblast，CEF）细胞为滋养层，进行体外培养，利用形态法、碱性磷酸酶（alkaline phosphatase，AKP）染色和胚胎阶段特异性表面抗原（embryo specific surface antigen 1，SSEA-1）检测对获得的胚胎干细胞进行鉴定。结果表明，获得典型的呈巢状或岛状的cESCs克隆，细胞AKP染色呈蓝紫色，表明其具有较高的内源性AKP活性；SSEA-1鉴定结果呈阳性，显示cESCs克隆具有多能性。采用10^-5 mol/L RA诱导鸡胚胎干细胞向雄性生殖细胞分化，镜下观察细胞形态变化，分别于诱导第0、2、4、6、8、10天提取细胞总RNA，反转录成cDNA，用于实时荧光定量PCR检测生殖细胞标志基因的表达。结果表明，在此诱导过程中，作为胚胎干细胞标志基因Nanog、Sox2表达量持续显著下降，而生殖细胞特异性基因Dazl、Stra8、c-kit、integrin α6表达量呈持续上升趋势；免疫细胞化学检测可观察到特异基因相关蛋白的阳性克隆。本研究成功分离出cESCs，可体外培养并保持未分化状态及多能性。10^-5 mol/L RA能够促进cESCs向雄性生殖细胞方向分化，可以引起生殖细胞相应基因的表达，为进一步研究雄性生殖细胞的形成和调控机制提供参考。

关键词: 鸡胚胎干细胞; 雄性生殖细胞; 体外培养; 性别鉴定; 视黄酸(RA); 诱导分化

Abstract: Embryonic stem cells (ESCs) differentiate into germ cells in vitro can be used to treat infertility and provide the best model for revealing the molecular mechanism of species from generation to generation.The study aimed to discuss the effect of retinoic acid (RA) on the differentiation of cESCs into male germ cells.ESCs were isolated from the X-stage chicken embryos of fresh breeding eggs by trypsin digestion,and then cultured in vitro with the chicken embryo fibroblast (CEF) cells as trophoblast.The morphogenesis,alkaline phosphatase(AKP) staining and SSEA-1 detection were used to identified the obtained ESCs.The results showed that typical cESCs clones were obtained as nests or islands,and the AKP cells were blue-purple,indicating high endogenous AKP activity.The result of SSEA-1 was positive,indicating the pluripotency of cESCs cloning.10^-5 mol/L RA was used to induce cESCs to differentiate into male germ cells (MGCs),the cell morphological changes were observed by microscopic,and total RNA was extracted at 0,2,4,6,8 and 10 day of induction cells,respectively,then the RNA was reverse transcribed into cDNA for Real-time quantitative PCR detection of germ cell marker genes expression.The results showed that ESCs marker genes Nanog,Sox2 expression decreased significantly in the induction,while the expression levels of germ cell-specific genes Dazl,Stra8,c-kit and integrin α6 all showed continuous upward trend.The specific gene-related proteins were observed of positive clones by immunocytochemistry test.In this study,cESCs were successfully isolated,which could be cultured in vitro and remained undifferentiated and pluripotent.10^-5 mol/L RA could induce the expression of corresponding genes in germ cells and promote the differentiation of cESCs into MGCs,laying a theoretical foundation for further study on formation and regulation mechanism of MGCs.

Key words: chicken embryonic stem cell; male germ cell; in vitro culture; sex determination; retinoic acid (RA); induced differentiation

中图分类号:

Q254

李欣, 赵中利, 吕阳, 柳俭强, 魏天, 金海国, 于永生. 视黄酸诱导鸡胚胎干细胞向雄性生殖细胞分化的研究[J]. 中国畜牧兽医, 2020, 47(7): 2142-2149.

LI Xin, ZHAO Zhongli, LYU Yang, LIU Jianqiang, WEI Tian, JIN Haiguo, YU Yongsheng. Study on Differentiation of Chicken Embryonic Stem Cells to Male Germ Cells by RA[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2142-2149.

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