蒙古马睾丸支持细胞的体外分离培养与鉴定

为研究一种简单快速分离蒙古马睾丸支持细胞并保证其活性的基本方法，本试验采集2岁蒙古马睾丸组织于低温环境下进行机械分离，将1~3 g睾丸组织剪碎，采用重力沉淀法去除游离的红细胞和间质细胞；使用0.1%胶原酶Ⅳ和0.25%胰蛋白酶+EDTA逐步进行组织消化，并用含有10%胎牛血清的培养基进行细胞培养；在接种后采用差异贴壁法分离纯化支持细胞，使用Tris-HCl低渗法去除杂质细胞，并采用碱性磷酸酶（AKP）染色、HE染色、实时荧光定量PCR方法进行鉴定。结果显示，支持细胞贴壁性能较强，培养24 h后大多数细胞开始贴壁，细胞形状呈椭圆形；培养48 h后胞质增多，折光性变强；培养3~4 d细胞胞质展开紧密连接，此时细胞呈三角形，有明显的核仁，培养6~7 d细胞生长状态进入稳定期。实时荧光定量PCR结果显示，GATA4和GDNF基因在培养细胞中极显著表达（P<0.01），AKP染色支持细胞呈阴性表达，表明分离培养的细胞确为支持细胞。本试验使用机械分离法与两步酶消化法处理组织，可快速高效地获得睾丸支持细胞，成功构建了蒙古马睾丸支持细胞体外培养方法。

Isolation,Culture and Identification of Testis Sertoli Cells in Mongolian Horses in vitro

To study a simple and rapid separation of testis Sertoli cells in Mongolian horses and ensure their activity,the testis tissue of two-year-old Mongolian horses was mechanically isolated under a low temperature environment in this experiment,and 1-3 g of testis tissue was chopped,the free red blood cells and interstitial cells were removed by gravity precipitation method.0.1% collagenase Ⅳ and 0.25% trypsin-EDTA were used for tissue digestion, and cells were cultured in medium containing 10% fetal bovine serum.After inoculation,the purified Sertoli cells were isolated and purified by differential sticking method,and the impurity cells were removed by Tris-HCl infiltration method,and were identified by alkaline phosphatase (AKP) staining,HE staining and Real-time quantitative PCR.The results showed that most of the cells began to adhere to the wall after culture 24 h,and the shape of the cells was oval.After culture 48 h,the cytoplasm increased and the refraction became stronger.After culture 3-4 d,the cytoplasm of the cells expanded into tight junction.At this time,the cells were triangular with obvious nucleoli.After culture 6-7 d,the cells entered the stable phase.Real-time quantitative PCR results showed that the expression of GATA4 and GDNF genes were extremely significantly expressed in cultured cells (P<0.01).AKP staining supported the negative expression in Sertoli cells,indicating that the isolated cultured cells were indeed Sertoli cells.In this experiment,tissue was treated by mechanical separation and two-step enzyme digestion,and testicular support cells were obtained quickly and efficiently,the method of culturing Sertoli cells in Mongolian horses testis in vitro was successfully constructed.