本文關(guān)鍵詞：DNAJB11促進(jìn)卵巢顆粒細(xì)胞中FOXL2誘導(dǎo)的雌激素合成　出處：《醫(yī)學(xué)研究生學(xué)報》2017年10期 　論文類型：期刊論文

  更多相關(guān)文章： DNAJB 轉(zhuǎn)錄因子FOXL 卵巢顆粒細(xì)胞 雌激素合成

【摘要】：目的轉(zhuǎn)錄因子FOXL2是顆粒細(xì)胞雌激素合成與顆粒細(xì)胞功能維持的關(guān)鍵調(diào)控分子,但目前尚不清楚FOXL2蛋白的表達(dá)及功能調(diào)控機制。文章旨在鑒定調(diào)控FOXL2蛋白功能的新分子。方法通過分離小鼠卵巢組織進(jìn)行免疫組化染色檢測DNAJB11的表達(dá)定位情況,含有全長DNAJB11基因c DNA序列(Ad-DNAJB11-Flag和Ad-Flag-DNAJB11)的腺病毒載體按Ad Max系統(tǒng)(Microbix)方法進(jìn)行病毒顆粒的包裝。按NE-PERTMNuclear and Cytoplasmic抽提試劑盒所述方法進(jìn)行細(xì)胞核質(zhì)蛋白的分離。KGN細(xì)胞感染不同濃度腺病毒(Ad-DNAJB11和Ad-Lac Z),測量波長450 nm的吸光度值,進(jìn)行細(xì)胞增殖檢測。采用Access Immunoassay System 2化學(xué)發(fā)光自動檢測系統(tǒng)(Beckman Coulter)檢測細(xì)胞培養(yǎng)上清中雌二醇的濃度。采用PCR方法擴增含有人FOXL2和人DNAJB 11基因的全長c DNA序列,并分別亞克隆至p CS2-6XMT載體(Myc-FOXL2)和p FLAG-CMV2載體(Flag-DNAJB11)中。采用Lipofectamine 2000轉(zhuǎn)染試劑將Myc-FOXL2和Flag-DNAJB11表達(dá)質(zhì)粒瞬時轉(zhuǎn)染至細(xì)胞HEK-293細(xì)胞中。收集HEK-293細(xì)胞或FSH處理的KGN細(xì)胞的裂解液,進(jìn)行FOXL2和DNAJB11的免疫共沉淀實驗。通過細(xì)胞免疫熒光染色和熒光素酶報告基因等實驗檢測內(nèi)質(zhì)網(wǎng)Hsp40/Dna J家族成員DNAJB11調(diào)控顆粒細(xì)胞的雌激素水平。結(jié)果 Western blot表明DNAJB11蛋白在人卵癌顆粒細(xì)胞KGN、小鼠原代卵巢顆粒細(xì)胞m GC以及小鼠卵巢中高表達(dá),免疫組化染色結(jié)果亦證實DNAJB11蛋白表達(dá)于小鼠卵巢的卵母細(xì)胞質(zhì)中,并在竇前卵泡、排卵前卵泡的顆粒細(xì)胞中持續(xù)表達(dá)。免疫熒光染色和Western blot分析亦表明外源性激素FSH可以誘導(dǎo)KGN細(xì)胞中內(nèi)源性DNAJB11蛋白從內(nèi)質(zhì)網(wǎng)轉(zhuǎn)移到細(xì)胞核中。腺病毒介導(dǎo)的DNAJB11-Flag過表達(dá)定位于細(xì)胞內(nèi)質(zhì)網(wǎng)中,但當(dāng)Flag標(biāo)簽阻斷DNAJB11信號肽功能時,外源FlagDNAJB11過表達(dá)則定位于顆粒細(xì)胞的細(xì)胞核,內(nèi)質(zhì)網(wǎng)中DNAJB11-Flag蛋白高表達(dá)以濃度依賴的方式減少KGN細(xì)胞中雌二醇的合成,與Ad-Lac Z(MOI=50)比較,Ad-DNAJB11-Flag(MOI=50)減少,KGN細(xì)胞中雌二醇的合成[(10 749.0±801.7)pg/m L vs(7 903.0±409.5)pg/m L,P0.01],而細(xì)胞核中高表達(dá)Flag-DNAJB11后則刺激KGN細(xì)胞中雌二醇的生成,與Ad-Lac Z(MOI=50)比較,Ad-DNAJB11-Flag(MOI=50)刺激雌二醇的生成[(10 749.0±801.7)pg/m L vs(14 217.0±1218.0)pg/m L,P0.01]。免疫共沉淀實驗表明當(dāng)Myc-tagged FOXL2與Flag-tagged DNAJB11分別共轉(zhuǎn)染HEK293細(xì)胞,FOXL2與DNAJB11可以相互免疫共沉淀對方,熒光素酶報告基因?qū)嶒炦M(jìn)一步表明細(xì)胞核中表達(dá)的DNAJB11顯著增強FOXL2介導(dǎo)的Cyp19A1啟動子活性和Cyp19A1蛋白表達(dá)。結(jié)論 DNAJB11是轉(zhuǎn)錄因子FOXL2新的結(jié)合分子并調(diào)控FOXL2蛋白的穩(wěn)定性與轉(zhuǎn)錄活性。
[Abstract]:Objective transcription factor FOXL2 is a key regulator of estrogen synthesis and granulosa cell function maintenance in granulosa cells, but the expression and functional regulation mechanism of FOXL2 protein is not yet clear. The aim of this article is to identify new molecules that regulate the function of FOXL2 protein. Methods the expression and location of DNAJB11 were detected by immunohistochemical staining of mouse ovarian tissue. The adenovirus vector containing full-length DNAJB11 gene C DNA sequence (Ad-DNAJB11-Flag and Ad-Flag-DNAJB11) was packaged according to Ad Max system (Microbix) method. The cell nuclear protein was separated according to the method described by NE-PERTMNuclear and Cytoplasmic extraction kit. KGN cells infected different concentrations of adenovirus (Ad-DNAJB11 and Ad-Lac Z), measured the absorbance value of 450 nm at wavelength, and detected the cell proliferation. The Access Immunoassay System 2 chemiluminescence automatic detection system (Beckman Coulter) was used to detect the concentration of estradiol in cell culture supernatant. The full-length C DNA sequence containing human FOXL2 and human DNAJB 11 gene was amplified by PCR and subcloned into P CS2-6XMT carrier (Myc-FOXL2) and P FLAG-CMV2 carrier (Flag-DNAJB11), respectively. Myc-FOXL2 and Flag-DNAJB11 expression plasmids were transiently transfected into cell HEK-293 cells by Lipofectamine 2000 transfection reagent. The lysate of KGN cells treated with HEK-293 or FSH was collected and the immunoprecipitation experiment of FOXL2 and DNAJB11 was carried out. The level of estrogen in granulosa cells regulated by Hsp40/Dna J family member DNAJB11 was detected by immunofluorescence staining and luciferase reporter assay. The results of Western blot showed that the high expression of DNAJB11 protein in primary human cancer cells, KGN egg granules of mouse granulosa cells of M and GC in mouse ovary, immunohistochemical staining also confirmed the expression of DNAJB11 protein in the mouse ovary in oocytes, granulosa cells and follicular, expressed in preantral preovulatory follicles in. Immunofluorescence staining and Western blot analysis also showed that exogenous FSH could induce endogenous DNAJB11 proteins in KGN cells to transfer from the endoplasmic reticulum to the nucleus. Adenovirus mediated overexpression of DNAJB11-Flag located in the endoplasmic reticulum, but when the Flag label blocking DNAJB11 signal peptide function, overexpression of FlagDNAJB11 is localized to the granule cell nucleus, endoplasmic reticulum in the high expression of DNAJB11-Flag protein in a concentration dependent manner to reduce the synthesis of estradiol in KGN cells, Ad-Lac and Z (MOI=50), Ad-DNAJB11-Flag (MOI=50) reduced synthesis of estradiol in KGN cells (10749 + 801.7) pg/m L vs (7903 + 409.5) pg/m, L, P0.01], and the nucleus in the high expression of Flag-DNAJB11 after the stimulation of production of estradiol and KGN cells, and Ad-Lac Z (MOI=50), Ad-DNAJB11-Flag (MOI=50 estradiol stimulates) [(10749 + 801.7) pg/m L vs (14217 + 1218) pg/m L, P0.01]. The experimental results show that when Myc-tagged FOXL2 and Flag-tagged DNAJB11 were co transfected into HEK293 cells by immunoprecipitation, FOXL2 and DNAJB11 can mutually co immunoprecipitation each other, luciferase reporter assays indicated that the nuclear expression of DNAJB11 significantly enhanced FOXL2 mediated by Cyp19A1 promoter activity and Cyp19A1 protein expression. Conclusion DNAJB11 is a new binding molecule of the transcription factor FOXL2 and regulates the stability and transcriptional activity of FOXL2 protein.
【作者單位】： 南京農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院;南京大學(xué)附屬鼓樓醫(yī)院生殖中心;
【基金】：國家自然科學(xué)基金(81370683,81571402) 江蘇省“十三五科教強衛(wèi)工程”醫(yī)學(xué)重點人才項目(ZDRCA2016070)
【分類號】：R339.22
【正文快照】： DNAJB11 promotes the synthesis of FOXL2-induced estradiol in ovarian granulosa cellsMAO Yan1,YAN Qiang2,ZHANG Chun-xue2,ZHEN Xin2,CAO Rui-bing1,YAN Gui-jun2(1.College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China;2.R

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