蒙古马睾丸支持细胞的体外分离培养与鉴定

doi:10.16431/j.cnki.1671-7236.2020.09.007

Abstract

Abstract: To study a simple and rapid separation of testis Sertoli cells in Mongolian horses and ensure their activity,the testis tissue of two-year-old Mongolian horses was mechanically isolated under a low temperature environment in this experiment,and 1-3 g of testis tissue was chopped,the free red blood cells and interstitial cells were removed by gravity precipitation method.0.1% collagenase Ⅳ and 0.25% trypsin-EDTA were used for tissue digestion, and cells were cultured in medium containing 10% fetal bovine serum.After inoculation,the purified Sertoli cells were isolated and purified by differential sticking method,and the impurity cells were removed by Tris-HCl infiltration method,and were identified by alkaline phosphatase (AKP) staining,HE staining and Real-time quantitative PCR.The results showed that most of the cells began to adhere to the wall after culture 24 h,and the shape of the cells was oval.After culture 48 h,the cytoplasm increased and the refraction became stronger.After culture 3-4 d,the cytoplasm of the cells expanded into tight junction.At this time,the cells were triangular with obvious nucleoli.After culture 6-7 d,the cells entered the stable phase.Real-time quantitative PCR results showed that the expression of GATA4 and GDNF genes were extremely significantly expressed in cultured cells (P<0.01).AKP staining supported the negative expression in Sertoli cells,indicating that the isolated cultured cells were indeed Sertoli cells.In this experiment,tissue was treated by mechanical separation and two-step enzyme digestion,and testicular support cells were obtained quickly and efficiently,the method of culturing Sertoli cells in Mongolian horses testis in vitro was successfully constructed.

Key words: Sertoli cells; Mongolian horses; isolation; in vitro culture

CLC Number:

S852.61⁺1

SONG Lianjie, CUI Yingying, ZHAO Yiping, BAI Dongyi, REN Xiujuan, TE Rigele, DUGARJAVIIN Manglai, LI Bei. Isolation,Culture and Identification of Testis Sertoli Cells in Mongolian Horses in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2751-2758.

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Isolation,Culture and Identification of Testis Sertoli Cells in Mongolian Horses in vitro

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