鸡胚睾丸支持细胞的分离、纯化与鉴定

Isolation, Purification and Characterization of Sertoli Cells from Embryonic Chickens

以18天鸡胚睾丸为实验材料,经胶原酶和胰蛋白酶两步酶消化法得到生精上皮细胞悬液。在原代培养过程中经差异贴壁和低渗处理后,获得了纯度约为90%的单层支持细胞。对纯化的培养物染色鉴定,结果显示支持细胞为碱性磷酸酶(AKP)阴性,而混杂在其中的另一种体细胞管周细胞为AKP阳性;油红O染色显示,支持细胞胞质内含有大量的脂肪滴,核内见双极小体;吖啶橙染色证实支持细胞富含RNA;罗丹明123染色显示支持细胞富含线粒体;Hoechst33342染色显示支持细胞细胞核呈长卵圆形,核的长轴与细胞长轴平行;免疫荧光染色显示支持细胞胞质中表达波形蛋白。建立了一套简单、易行的鸡胚睾丸支持细胞分离纯化与鉴定方法。

Testes from 18-day-old embryonic chickens were used to isolate seminiferous epithelial cells by sequential two-step enzyme digestion with collagenase and trypsin. During primary culture, highly purified monolayers containing about 90% sertoli cells can be successfully achieved by differential plating and hypotonic treatment. The sertoli cell-enriched cultures were then characterized by staining. The results showed that, no alkaline phosphatase （AKP） staining were found in sertoli cells itself, while another somatic cells, peritubular myoid cells were AKP positive, lipid droplet formation in the cytoplasm and bipolar corpuscula in nucleus were clearly detected after oil red O staining. Acridine orange staining demonstrated that sertoli cells were rich in RNA. Rhodamine 123 staining showed that sertoli cells were rich in mitochondria. Hoechst 33342 staining showed that sertoli cells had a long elliptical shaped nucleus and the long axis of nucleus oriented parallel to the long axis of the cell. Immunofluorescent staining showed that vimentin was expressed in sertoli cell cytoplasm. A simple and reliable method for isolation, purification and characterization of sertoli cells from embryonic chickens has been established.