Culture of Yak (Bos grunniens) Granulosa Cells In vitro and Effects of FGF-10 on Its DBI Expression
WANG Qiang-Long, PAN Yang-Yang, GAO Ze-Chuan, ZHANG Tong-Xiang, CUI Yan, XU Geng-Quan, WANG Li-Bin, FAN Jiang-Feng, YU Si-Jiu*
Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep & Goat/College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Diazepam binding inhibitor (DBI) is closely related to cellular fatty acid metabolism, and fatty acids are involved in oocyte maturation and follicle development. In order to explore the potential biological role of maternal secretory factors mediated by DBI in regulating ovarian development and oocyte maturation of yaks (Bos grunniens). Through the isolation and establishment of yak granulosa cells in vitro culture system, different concentrations of exogenous fibroblast growth factor-10 (FGF-10) (0, 20, 50, 100, 150, 200 ng/mL) were added in the process of in vitro culture in different time (12, 24 h). qPCR , Western blot (WB) and immunofluorescence (IF) assays were used to detect the effect of different concentrations of FGF-10 on the expression of DBI in granulosa cells from gene and protein levels. The isolation and culture system of primary yak granulosa cells was successfully established as follows: The granulosa cell inoculation density was 4×105/mL, DMEM/F12 medium+12% FBS+100 U/mL streptomycin+100 U/mL penicillin, changed the liquid once in 36 h, and the purity of the cells was as high as 95%. After treated with 100 ng/mL FGF-10 for 12 h, the expression level of DBI gene and protein was the highest, and the expression level of DBI was decreased in each treatment group at 24 h. Immunofluorescence showed that DBI protein could be expressed in both the nucleus and cytoplasm of granulosa cells in each treatment group. The results showed that FGF-10 was involved in regulating the expression of DBI in yak granulocytes in a concentration-dependent and time-difference manner, which provides a theoretical basis for further exploring the multiple biological mechanisms of maternal cytokines regulating oocyte maturation.
王强龙, 潘阳阳, 高泽川, 张同享, 崔燕, 徐庚全, 王立斌, 樊江峰, 余四九. 牦牛颗粒细胞体外培养及FGF-10对其DBI表达的影响[J]. 农业生物技术学报, 2022, 30(2): 316-324.
WANG Qiang-Long, PAN Yang-Yang, GAO Ze-Chuan, ZHANG Tong-Xiang, CUI Yan, XU Geng-Quan, WANG Li-Bin, FAN Jiang-Feng, YU Si-Jiu. Culture of Yak (Bos grunniens) Granulosa Cells In vitro and Effects of FGF-10 on Its DBI Expression. 农业生物技术学报, 2022, 30(2): 316-324.
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