从江香猪睾丸支持细胞分离培养与鉴定

Isolation, culture and identification of Sertoli cells from the Congjiang Xiang pig testis

【目的】建立一种高效制备从江香猪睾丸支持细胞(Sertoli cells, SCs)体外分离培养方法,为后续香猪雄性繁殖研究提供细胞材料,为同类小型香猪支持细胞的分离培养提供参考。【方法】采用0.1%Ⅳ型胶原酶和0.25%胰酶-EDTA组合酶消化法消化睾丸组织,差速贴壁法纯化支持细胞,使用含10%胎牛血清完全培养基培养支持细胞,观察其形态特征并记录生长曲线,经苏木精—伊红(HE)染色、RT-PCR及免疫荧光染色进行支持细胞的鉴定。【结果】组合酶法消化后可获得大量生精上皮细胞混悬液,刚分离的支持细胞呈圆形或椭圆形,分离培养5~6 h后SCs贴壁,贴壁后细胞形态呈梭形或不规则形,培养3~4 d后细胞之间呈紧密连接,可清晰观察到细胞核及细胞质中的颗粒状物质或小空泡;SCs分离后1~2 d增殖速度缓慢,培养3~6 d细胞增殖速度加快,活性增强,进入对数生长期,培养7~8 d后细胞增殖速率下降,原代支持细胞生长曲线呈S形;HE染色显示SCs细胞核为深紫色,细胞质呈淡红色;RT-PCR结果显示支持细胞特异性基因WT1、SOX9均表达;免疫荧光染色结果显示特异性基因GATA-4抗体免疫阳性率达90%以上。【结论】成功建立从江香猪睾丸支持细胞的原代培养方法。

【Objective】This study aimed to establish an effective method for isolation and culture of testicular Sertoli cells(SCs) from the Congjiang Xiang pig in vitro, provide cell material for the following reproduction of male Xiang pig, and provide reference for the isolation and cultivation of SCs from same type of Xiang pig.【Method】Using the combined enzyme digestion method of 0.1% collagenase Ⅳ and 0.25% trypsin-EDTA, testicular tissues were digested to isolate SCs. SCs were purified by differential adhesion method and cultured in the complete medium containing 10% fetal bovine serum. The morphological characteristics and growth curve of SCs from the Congjiang Xiang pig were observed, and SCs was identified by hematoxylin-eosin staining, RT-PCR and immunofluorescence staining. Identifying SCs by hematoxylin-eosin staining, RT-PCR and immunofluorescence staining.【Result】A large number of spermatogenic epithelial cell suspensions could be obtained after combined enzyme digestion. The isolated SCs were round or oval, and the adherent cells after culturing 5-6 hours were triangular or irregular. SCs showed tight junction after 3-4 days, and subsequently, the cell proliferation rate decreased after 7-8 days and the growth curve of primary Sertoli cells was S-shaped. HE staining showed that the nucleus of SCs was dark purple and the cytoplasm was light red;RT-PCR showed that Sertoli cell specific genes WT1 and SOX9 were expressed, and the positive rate of GATA-4(a SC-specific maker) was more than 90% in immunofluorescence staining.【Conclusion】Aculture method of testicular SCs of the Congjiang Xiang pig in vitro is successfully established.