ES细胞体外定向分化为睾丸间质样细胞模型的建立

Construction of directional differentiation model from mouse embryonic stem cells to leydig-like cells in vitro

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。

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.