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浙江大学学报（医学版）
浙江大学学报（医学版）  2012, Vol. 41 Issue (4): 386-392    DOI: 10.3785/j.issn.1008-9292.2012.04.006
专题报道     
ES细胞体外定向分化为睾丸间质样细胞模型的建立
张莹莹1，黄亚东2，葛仁山3，朱丹雁1
1．浙江大学药学院 药理毒理与生化药学研究所，浙江 杭州 310058； 2.暨南大学生命科学技术学院，广东 广州 510632； 3.美国洛克菲勒大学，美国人口理事会，生物医学研究中心，纽约 10065
Construction of directional differentiation model from mouse embryonic stem cells to leydig-like cells in vitro
ZHANG Ying-ying1, HUANG Ya-dong2, GE Ren-shan3, ZHU Dan-yan1
1. Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou 310058, China; 2.College of Life Science and Technology, Jinan University, Guangzhou 510632, China; 3.Rockefeller University, Population Council, Center for Biomedical Research, New York 10065, USA
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摘要： 目的：建立小鼠胚胎干（embryonic stem，ES）细胞体外分化为睾丸间质样细胞（leydig-like cells）的模型。
方法：采用脂质体转染方法将含类固醇生成因子1（steroidogenic factor 1，SF-1）基因片断的质粒转入小鼠ES-D3细胞，并在RA和8Br-cAMP作用下，体外诱导ES-D3细胞分化为睾丸间质样细胞，以空质粒转染组做阴性对照；通过光镜观察转染SF-1的ES-D3细胞定向分化为睾丸间质样细胞过程中的形态变化；用RT-PCR及Western Blot法检测分化过程中类固醇激素合成急性调节蛋白（StAR），胆固醇侧链裂解酶P450scc (CYP11A1)和3β-羟甾脱氢酶 1（3β-HSD1）表达情况；免疫荧光法检测睾丸间质样细胞特异性蛋白P450scc及3β-HSD1表达。
结果：ES-D3细胞转染SF-1质粒24 h，即检测到SF-1基因表达。转染成功的ES-D3细胞在RA和8Br-cAMP诱导分化48 h后出现睾丸间质样细胞。分化而来的睾丸间质样细胞胞体大而圆润，长有触角，呈纺锤形；且表达睾丸间质样细胞特异性蛋白P450scc和3β-HSD1，同时StAR表达显著增加，而对照组细胞未表达睾丸间质样细胞特异性标志物StAR、P450scc和3β-HSD1。
结论：通过对ES-D3细胞转染SF-1质粒并予以RA和8Br-cAMP诱导，成功建立ES-D3细胞定向分化为睾丸间质样细胞模型。
关键词： 胚胎干细胞;  类固醇生成因子1;  睾丸/细胞学;  间质细胞;  SF-1;  睾丸间质样细胞    
Abstract: Objective： To construct a directional differentiation model from mouse embryonic stem cells into leydig-like cells in vitro.
Methods： Mouse ES-D3 cells were transfected with plasmid containing steroidogenic factor 1 (SF-1) gene, then treated with RA and 8Br-cAMP, while the cells transfected with empty plasmid were used as the negative controls. The morphology of leydig-like cells differentiated from ES-D3 cells was observed with light microscopy. The expression levels of StAR, P450scc and 3β-HSD were detected by RT-PCR, Western Blot and fluorescence microscopy analysis in leydig-like cells derived from the ES cells.
Results： ES-D3 cells were transfected with plasmid containing SF-1 gene successfully, and SF-1 was expressed 24 h after transfection. The SF-1-transfected ES-D3 cells were induced by RA and 8Br-cAMP to differentiate into leydig-like cells. The differentiated cells showed spindle shape with tentacles, which expressed the specific protein marker for leydig cells 3β-HSD1 and P450scc. Meanwhile, in these leydig-like cells, the expression of StAR increased compared with control group. 3β-HSD1, P450scc and StAR were not detected in negative control group.
Conclusion： When the ES-D3 cells are transfected with SF-1 plasmid and then treated with RA and 8Br-cAMP, the cells are able to differentiate into leydig-like cells, indicating that the model of directional differentiation of ES cells into leydig-like cells has been constructed successfully.
Key words: Embryonic stem cells    Steroidogenic factor 1    Testis/cytol    Stromal cells    SF-1    Leydig-like cells
出版日期: 2012-07-25
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张莹莹;黄亚东;葛仁山;朱丹雁. ES细胞体外定向分化为睾丸间质样细胞模型的建立[J]. 浙江大学学报（医学版）, 2012, 41(4): 386-392.

链接本文:

https://www.zjujournals.com/med/CN/Y2012/V41/I4/386

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