Zhang Ying-ying, Huang Ya-dong, Ge Ren-shan, Zhu Dan-yan
Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou 310058, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2012 Jul;41(4):386-92.
To construct a directional differentiation model from mouse embryonic stem cells into leydig-like cells in vitro.
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.
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.
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.
构建小鼠胚胎干细胞体外定向分化为类睾丸间质细胞的模型。
用含类固醇生成因子1（SF-1）基因的质粒转染小鼠ES-D3细胞，然后用视黄酸（RA）和8-溴环磷酸腺苷（8Br-cAMP）处理，以转染空质粒的细胞作为阴性对照。用光镜观察ES-D3细胞分化而来的类睾丸间质细胞的形态。通过逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（Western Blot）和荧光显微镜分析检测ES细胞来源的类睾丸间质细胞中类固醇生成急性调节蛋白（StAR）、细胞色素P450侧链裂解酶（P450scc）和3β-羟基类固醇脱氢酶（3β-HSD）的表达水平。
成功用含SF-1基因的质粒转染ES-D3细胞，转染后24小时SF-1表达。转染SF-1的ES-D3细胞经RA和8Br-cAMP诱导分化为类睾丸间质细胞。分化后的细胞呈带有触须的纺锤形，表达睾丸间质细胞特异性蛋白标志物3β-HSD1和P450scc。同时，与对照组相比，这些类睾丸间质细胞中StAR的表达增加。阴性对照组未检测到3β-HSD1、P450scc和StAR。
当ES-D3细胞用SF-1质粒转染后再用RA和8Br-cAMP处理时，细胞能够分化为类睾丸间质细胞，表明ES细胞向类睾丸间质细胞定向分化的模型已成功构建。
