牦牛颗粒细胞体外培养及FGF-10对其DBI表达的影响  

Culture of Yak(Bos grunniens)Granulosa Cells In vitro and Effects of FGF-10 on Its DBI Expression

作　　者：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 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)

机构地区：[1]甘肃农业大学动物医学院/甘肃省牛羊胚胎工程技术研究中心,兰州730070

出　　处：《农业生物技术学报》2022年第2期316-324,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972760);甘肃省杰出青年基金(20JR10RA561);甘肃省教育厅产业支撑引导项目(2019C-03);甘肃省教育厅创新能力提升项目(2019B-081)。

摘　　要：地西泮结合抑制因子(diazepam binding inhibitor,DBI)与细胞脂肪酸代谢密切相关,而脂肪酸参与卵母细胞成熟和卵泡发育。为探索其介导的母源分泌因子调控牦牛(Bos grunniens)卵巢发育和卵母细胞成熟的潜在生物学作用,本研究通过分离与建立牦牛颗粒细胞体外培养体系,体外培养过程中加入不同浓度外源性成纤维细胞生长因子10(fibroblast growth factor-10,FGF-10)(0,20,50,100,150,200 ng/mL)作用不同时间后(12,24 h)。采用qPCR、蛋白免疫印迹技术(Western blot,WB)和免疫荧光技术(immunofluorescence,IF)从基因和蛋白水平检测不同浓度FGF-10对颗粒细胞DBI表达的影响。研究成功建立了原代牦牛颗粒细胞分离与培养体系,具体为:颗粒细胞接种密度4×10^(5)个/mL,DMEM/F12培养基+12%FBS+100 U/mL链霉素+100 U/mL青霉素,36 h换液1次,细胞的纯度高达95%;100 ng/mL FGF-10作用牦牛颗粒细胞12 h时,DBI基因和蛋白表达水平最高,24 h各处理组DBI表达水平降低,免疫荧光显示各处理组颗粒细胞的细胞核和细胞质均可表达DBI蛋白。结果表明FGF-10参与调控牦牛颗粒细胞DBI的表达,并且具有浓度依赖性和时间差异性。本研究为进一步探索母源细胞因子调控卵母细胞成熟的多重生物学机制提供了理论依据。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 4×10^(5)/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 timedifference manner,which provides a theoretical basis for further exploring the multiple biological mechanisms of maternal cytokines regulating oocyte maturation.

关 键 词：牦牛 颗粒细胞 培养 地西泮结合抑制因子(DBI) 成纤维细胞生长因子10(FGF-10) 

分 类 号：S831.2[农业科学—畜牧学]