新生牛睾丸未成熟支持细胞的分离培养与鉴定

Isolation, culture and identification of immature Sertoli cells from neonatal bovine testis

为了探究中国西门塔尔牛新生牛睾丸未成熟支持细胞的体外培养特性,为细胞水平研究与繁殖性状相关候选基因奠定基础。以新生牛曲细精管为试验材料,利用组合酶消化法将其解离成单细胞悬液,采用差速贴壁法纯化未成熟支持细胞后进行体外培养与鉴定。结果表明:福尔根染色结果可见所分离的细胞具有明显的双极小体;RT-PCR检测结果显示,所分离的细胞中支持细胞标志基因SCF、GDNF和BMP4均有表达;免疫荧光染色结果表明,支持细胞特异性标志蛋白GATA4和FASL以及未成熟支持细胞标志蛋白KRT-18均有表达,纯度较高。本试验成功分离了中国西门塔尔牛未成熟支持细胞,为新生牛睾丸未成熟支持细胞的分离培养提供了一种高效的实验方法,同时为后续开展未成熟支持细胞调控雄性生殖过程的机制研究提供了试验依据。

In order to explore the in vitro culture characteristics of immature Sertoli cells of Simmental newborn bovine testis, the seminiferous tubules of newborn cattle were used as experimental materials, which were dissociated into single cell suspension by combined enzyme digestion method, and the immature Sertoli cells were purified by differential adhesion method for in vitro culture and identification.The results showed that the isolated cells had obvious bipolar bodies by Folgen staining;the results of RT-PCR showed that marker genes SCF,GDNF and BMP4 of the Sertoli cell were expressed in the isolated cells;the results of immunofluorescence staining showed that the specific marker proteins GATA4 and FasL of the Sertoli cell and the marker protein krt-18 of immature Sertoli cell were expressed with high purity.The above results show that immature Sertoli cells of Simmental cattle were successfully isolated in this experiment, which provides an efficient experimental method for the isolation and culture of immature Sertoli cells of newborn cattle testis, and provides experimental materials for the subsequent research of the mechanism of immature Sertoli cells regulating male reproductive process.