Culture and identification of testis Sertoli cells in calf in vitro | 新生牛睾丸支持细胞的体外培养及鉴定分析
2013
Yu Lei, Northeast Agricultural University, Harbin (China), College of Animal Science and Technology | Zheng Peng, Northeast Agricultural University, Harbin (China), College of Animal Science and Technology | Rong Enguang, Northeast Agricultural University, Harbin (China), College of Animal Science and Technology
Chinese. 为探究支持细胞的体外培养特性，以新生牛睾丸组织为试验材料，利用组合酶消化法将其解离成单细胞悬液，并采用差速贴壁法纯化支持细胞后进行体外培养与鉴定。结果表明：支持细胞体外培养能够传至第20代，并且随着传代次数的增加，支持细胞的增殖速度越来越慢；添加2%血清浓度的DMEM/F-12的培养基即可用于支持细胞的体外培养；免疫组化染色显示，所培养的细胞内波形蛋白呈阳性；RT-PCR检测结果可见支持细胞标志基因SCF和GDNF的表达；第15代的支持细胞内含有正常的二倍体核型。
Show more [+] Less [-]English. In order to explore the cultural characteristics of sertoli cells in vitro, calf testis were used in the present study. The method of collagenase and typin was sequentially performed to separate sertoli cells, and then the differential adhesion method was used to purify sertoli cells. The sertoli cells obtained were cultured in vitro and identified. The results showed that the sertoli cells could be passaged to the 20th generation, and with the increase of the number of passages, cell proliferation ratio was getting slower and slower; DMEM/F-12 medium containing 2% fetal bovine serum could be used to culture sertoli cells in vitro; Immunohistochemical staining result indicated that sertoli cells expressed vimentin. The marker gene SCF and GDNF could be detected by RT-PCR analysis in sertoli cells. The 15th generation of sertoli cells contained normal diploid karyotype. This study not only identified calf sertoli cells effectively, but also laid foundation for the probe into the physiological function of sertoli cells cultured in vitro.
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