地西泮结合抑制剂在牦牛不同大小卵泡中的表达与定位

xpression and localization of DBI in yak follicles of different sizes

【目的】研究地西泮结合抑制剂(diazepam binding inhibitor, DBI)在牦牛不同大小卵泡中的表达水平与定位,探讨DBI蛋白在牦牛卵泡发育过程中的调控作用。【方法】采集牦牛卵巢组织,分离不同大小(直径<3 mm,直径3~8 mm,直径>8 mm)的卵泡。采用实时荧光定量方法(qRT-PCR)检测DBI基因mRNA在牦牛不同大小卵泡中的相对表达量,并利用蛋白免疫印迹技术(Western-blotting, WB)检测DBI蛋白的表达情况。采用免疫组织化学方法(immunohisto chemistry, IHC)定位DBI蛋白在雌性牦牛完整成熟卵泡中的分布情况。采用免疫荧光技术(immunofluorescence, IF)检测DBI在牦牛卵母细胞中的定位。【结果】DBI基因mRNA和DBI蛋白在牦牛不同大小卵泡中均表达,但表达水平存在显著差异(P<0.05),表达量随着牦牛卵泡直径的增大而显著上升。DBI蛋白在牦牛成熟卵泡中的颗粒细胞、卵丘细胞均有分布;在卵母细胞中主要定位于细胞质、细胞核。【结论】DBI在类固醇激素合成与卵母细胞成熟过程中具有重要作用。

【Objective】This study investigated the expression level and localization of diazepam binding inhibitor(DBI)in yak follicles of different sizes and investigated the regulatory role of DBI in follicular development.【Method】Yak ovarian tissues were collected and follicles of different sizes(diameter<3 mm,diameter 3-8 mm and diameter>8 mm)were isolated.The relative expression of DBI gene mRNA in follicles of different sizes was detected by quantitative real-time fluorescence(qRT-PCR),and the expre-ssion of DBI protein was detected by Western-blotting(WB).Immunohisto chemistry(IHC)was used to localize the distribution of DBI protein in intact mature follicles of female yaks.Immunofluorescence(IF)technique was used to detect the distribution of DBI in yak oocytes.【Result】DBI mRNA and protein were expressed in follicles of different sizes with significant differences in expression levels(P<0.05),and the expressions increased with the increase of follicle diameter in yak.DBI protein distributed in granulosa cells and cumulus cells,and it was mainly localized in the cytoplasm and nucleus of oocyte.【Conclusion】DBI played an important role in the synthesis of steroidogenic hormones and oocyte maturation.