奶山羊睾丸支持细胞分离培养与鉴定

Isolation,Culture and Identification of Sertoli Cells from Dairy Goat

本研究以2月龄莎能奶山羊为实验动物分离睾丸支持细胞(SCs),比较三种酶消化法(胰酶消化法、两步酶消化法和两步混合酶消化法)的分离效率,并采用形态学及分子生物学技术评估培养效果。结果显示,两步酶消化法和两步混合酶消化法获得的细胞数量以及细胞活率和增殖力均显著高于胰酶消化法。形态学观察两步酶消化法分离的细胞符合典型SCs形态,并能稳定传代至第5代。HE染色可观察到细胞核中的双极小体,油红O染色可见细胞核周围大小不一的脂滴。免疫荧光染色显示SCs特异蛋白WT1和Vimen-tin抗体免疫阳性率大于96%。超微结构观察发现细胞结构完整,细胞器丰富,细胞核呈不规则形状,符合SCs超微结构特征。综上,两步酶消化法操作简单、成本低,且分离的SCs数量多、活力好、纯度高,能在体外稳定培养,为最佳的奶山羊SCs分离方法,可为深入开展奶山羊雄性生殖机能研究提供良好的细胞学模型。

In this study,the Sertoli cells(SCs)were isolated from 2-month-old Shannon dairy goats,and the isolation efficiency of three enzyme digestion methods(trypsin digestion,two-step enzyme digestion,and two-step mixed enzyme digestion)were compared.The morphological and molecular biological techniques were used to characterize and evaluate the culture effects.The results showed that the number,viability and proliferation of cells obtained by two-step enzyme digestion and two-step mixed enzyme digestion were signifi-cantly higher than those obtained by pancreatic enzyme digestion.Morphological observation showed that the cells isolated by two-step enzyme digestion method had typical SCs morphology and could be stably passaged to the fifth generation.HE staining showed bipolar corpuscula in the nucleus,and oil red O staining showed lipid droplets with different sizes around the nucleus.Immunofluorescence staining showed that the positive rate of SCs specific protein WT1 and Vimentin antibodies was more than 96%.The ultrastructural observation showed that the cell structure was intact,the organelles were abundant,and the nucleus was irregular in shape,which was consistent with the ultrastructural characteristics of SCs.In conclusion,the two-step enzyme digestion method was simple,low-cost,and could isolate a large number of SCs with good viability and purity,which could be stably cultured in vitro,so it was the best method for isolating SCs from dairy goats,and could pro-vide a good cytological model for the in-depth study of male reproductive function of dairy goats.