外源添加17β-雌二醇对牦牛输卵管上皮细胞DBI表达的影响

Effects of exogenous 17β-estradiol on DBI expression in yak oviduct epithelial cells

地西泮结合抑制因子(diazepam binding inhibitor,DBI)在动物组织广泛表达,与细胞脂肪酸代谢密切相关,而脂肪酸参与早期胚胎发育能量供应。[目的]为研究其介导的母源分泌因子调控牦牛输卵管上皮细胞物质代谢和哺乳动物早期胚胎能量供给之间的潜在生物学作用。[方法]试验通过分离并建立牦牛输卵管上皮细胞体外培养体系,培养过程中添加不同浓度外源性17β-雌二醇(0、1×10^(-6)、1×10^(-7)、1×10^(-8)、1×10^(-9)、1×10^(-10) moL/L),作用不同时间后(0、6、12、24、48、72 h)。采用实时荧光定量PCR(quantitative real-time PCR,qPCR)和免疫荧光技术(immunofluorescence,IF)从基因水平分别检测不同浓度17β-雌二醇对输卵管上皮细胞DBI表达的影响。[结果]试验成功建立了原代牦牛输卵管上皮细胞分离与培养体系:输卵管上皮细胞接种密度4×10^(5)个/mL,DMEM/F12培养基+10%FBS+100 U/mL链霉素+100 U/mL青霉素,24 h换液1次,细胞的纯度高达90%以上;1×10^(-7)moL/L 17β-雌二醇作用牦牛输卵管上皮细胞6 h时,DBI基因表达水平最高,其它各处理组DBI表达水平降低,免疫荧光显示各处理组输卵管上皮细胞的细胞核和细胞质均可表达DBI蛋白。[结论]17β-雌二醇参与调控牦牛输卵管上皮细胞DBI的表达,并且具有剂量依赖性和时间差异性,为进一步探索母源细胞因子调控输卵管上皮细胞的多重生物学机制提供了关键科学突破点。

Diazepam binding inhibitor(DBI)is widely expressed in animal tissues,which is closely related to cellular fatty acid metabolism,and fatty acid is involved in energy supply for early embryonic development.[Objective]The aim of this study was to investigate the potential biological role of maternal secretory factor mediated in regulating material metabolism of yak oviduct epithelial cells and energy supply of early mammalian embryos.[Methods]In this study,yak oviduct epithelial cells were isolated and cultured in vitro.Exogenous 17β-estradiol(1×10^(-6),1×10^(-7),1×10^(-8),1×10^(-9),1×10^(-10)mol/L)was added into the cultured cells for different time(0,6,12,24,48,72 h).The effect of 17β-estradiol on DBI expression in oviduct epithelial cells was detected at gene levels by Quantitative real-time PCR(qPCR)and Immunofluorescence(IF).[Results]The isolation and culture system of primary yaks otubal epithelial cells was successfully established.The inoculation density of otubal epithelial cells was 4×105/mL,DMEM/F12 medium+10%FBS+100 U·mL^(-1) streptomycin+100 U·mL^(-1) penicillin,changing the liquid every 24 hours.And the purity of otubal epithelial cells was more than 90%.After the treatment with 1×10^(-7) mol/L 17β-estradiol for 6 h,the gene expression levels of DBI were the highest,while the expression levels of DBI in other treatment groups decreased.Immunofluorescence showed that DBI protein could be expressed in the cell nucleus and cytoplasm of tubal epithelial cells in all treatment groups.[Conclusion]17β-estradiol was involved in the regulation of DBI expression in yak oviduct epithelial cells in a dose-dependent and temporal variabilities,providing a key scientific breakthrough for further exploring the multiple biological mechanisms of maternal cytokines regulation of oviduct epithelial cells.