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

Abstract

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

CLC Number:

Q254

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

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