發(fā)布時(shí)間：2018-07-24 21:15

【摘要】：目的初步研究小鼠附睪Fkbp5(FK506 binding protein 5)基因?qū)π奂に氐捻憫?yīng)性以及相關(guān)的分子機(jī)制。方法建立小鼠去勢(shì)以及雄激素補(bǔ)充模型。采用RT-PCR以及RT-qPCR,檢測(cè)正常小鼠、去勢(shì)小鼠以及去勢(shì)后補(bǔ)充外源雄激素的小鼠附睪中Fkbp5基因的mRNA表達(dá)水平。搜索染色體免疫共沉淀-測(cè)序(chromatin immunoprecipitation-sequencing,ChIP-seq)數(shù)據(jù)建立的小鼠附睪全基因組雄激素受體(androgen receptor,AR)結(jié)合位點(diǎn)的數(shù)據(jù)庫(kù),尋找與Fkbp5基因相關(guān)聯(lián)的AR結(jié)合位點(diǎn)。利用AR抗體進(jìn)行染色體免疫共沉淀(chromatin immunoprecipitation,ChIP),采用ChIP-PCR以及ChIP-qPCR的方法,檢測(cè)Fkbp5基因相關(guān)聯(lián)的AR結(jié)合位點(diǎn)在體內(nèi)與AR的結(jié)合情況。結(jié)果去勢(shì)后,Fkbp5基因的表達(dá)顯著降低(P0.05);補(bǔ)加雄激素后,Fkbp5基因的表達(dá)又升至正常水平(P0.05)。從小鼠附睪AR結(jié)合位點(diǎn)的數(shù)據(jù)庫(kù)中鑒定到14個(gè)Fkbp5相關(guān)聯(lián)的AR結(jié)合位點(diǎn)。ChIP-PCR結(jié)果顯示,在正常生理狀態(tài)下,這14個(gè)結(jié)合位點(diǎn)均與AR結(jié)合。ChIP-qPCR結(jié)果顯示,去勢(shì)后,這14個(gè)AR結(jié)合位點(diǎn)的富集倍數(shù)均顯著下降(P0.05);而補(bǔ)加雄激素后,這些AR結(jié)合位點(diǎn)的富集倍數(shù)又顯著上升至生理水平(P0.05)。結(jié)論 Fkbp5在小鼠附睪內(nèi)的表達(dá)受雄激素的正調(diào)控,并且是AR的一個(gè)直接靶基因。該研究首次證實(shí)了Fkbp5的啟動(dòng)子以及上游增強(qiáng)子區(qū)域存在雄激素響應(yīng)元件,為探究雄激素/AR對(duì)Fkbp5的調(diào)控機(jī)制提供了新的視角。
[Abstract]:Objective to study the response of mouse epididymal Fkbp5 (FK506 binding protein 5) gene to androgen and its molecular mechanism. Methods A model of castration and androgen supplementation in mice was established. RT-PCR and RT-qPCR were used to detect the mRNA expression of Fkbp5 gene in epididymis of normal mice, ovariectomized mice and ovariectomized mice supplemented with exogenous androgen. To search the database of androgen receptor AR binding site in mouse epididymis genome based on chromatin immunoprecipitation-sequencing-sequenced data, and to search for the AR binding site associated with Fkbp5 gene. Chromatin immunoprecipitation chip (chip) was carried out with AR antibody. The AR binding sites associated with Fkbp5 gene were detected by ChIP-PCR and ChIP-qPCR in vivo. Results after castration, the expression of Fkbp5 gene decreased significantly (P0.05), and the expression of Fkbp5 gene increased to normal level after androgen supplementation (P0.05). Fourteen AR binding sites associated with Fkbp5 were identified from the mouse epididymal AR binding site database. ChIP-PCR results showed that under normal physiological conditions, these 14 binding sites were all associated with AR. ChIP-qPCR results showed that, after castration, all the 14 binding sites were associated with AR. The enrichment ratio of these 14 AR binding sites decreased significantly (P0.05), and the enrichment multiple of these AR binding sites increased significantly to physiological level after androgen supplementation (P0.05). Conclusion the expression of Fkbp5 in mouse epididymis is positively regulated by androgen and is a direct target gene of AR. This study confirmed for the first time the existence of androgen response elements in the promoter and upstream enhancer of Fkbp5, which provided a new perspective for exploring the regulation mechanism of androgen / AR on Fkbp5.
【作者單位】： 上海大學(xué)生命科學(xué)學(xué)院;上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院生殖醫(yī)學(xué)科;上海市輔助生殖與優(yōu)生重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家自然科學(xué)基金(81571435,81200468)資助
【分類(lèi)號(hào)】：Q492

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