發(fā)布時(shí)間：2018-02-26 09:39

  本文關(guān)鍵詞： 雌性小鼠 胚胎植入 蛻膜化 類固醇激素 顆粒細(xì)胞　出處：《西北農(nóng)林科技大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：Activating transcription factor 6(ATF6)是一個(gè)位于細(xì)胞內(nèi)質(zhì)網(wǎng)膜上的II型跨膜蛋白,屬于CREB轉(zhuǎn)錄因子家族成員。ATF6作為內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)轉(zhuǎn)導(dǎo)通路中的一個(gè)主要壓力感受器(Stress Sensor)蛋白,發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激時(shí),它能夠與相關(guān)的內(nèi)質(zhì)網(wǎng)應(yīng)激響應(yīng)基因元件協(xié)同而調(diào)節(jié)細(xì)胞中蛋白質(zhì)的合成與降解。ATF6廣泛表達(dá)于機(jī)體的多種組織、器官和細(xì)胞中,如胎盤、子宮的蛻膜組織、肝臟、心臟、胎兒成纖維細(xì)胞和RAW264.7巨噬細(xì)胞等。現(xiàn)有研究表明,內(nèi)質(zhì)網(wǎng)應(yīng)激及其信號(hào)轉(zhuǎn)導(dǎo)通路中的相關(guān)基因在哺乳動(dòng)物的早期胚胎植入、子宮內(nèi)膜基質(zhì)細(xì)胞蛻膜化和卵巢功能的維持與發(fā)揮等一系列生殖過程中發(fā)揮了重要的調(diào)節(jié)作用。因而,進(jìn)一步深入揭示ATF6及其介導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路在雌性哺乳動(dòng)物生殖過程中的作用,對(duì)于全面認(rèn)識(shí)哺乳動(dòng)物生殖調(diào)控的分子機(jī)制、探尋調(diào)控動(dòng)物繁殖的新靶點(diǎn)具有重要價(jià)值。本研究以小鼠為模型,應(yīng)用免疫組織化學(xué)染色、qRT-PCR、Western blotting、shRNA干擾、流式細(xì)胞術(shù)和TUNEL等技術(shù)和方法,對(duì)內(nèi)網(wǎng)應(yīng)激相關(guān)基因ATF6在小鼠胚胎植入時(shí)期的子宮和卵巢顆粒細(xì)胞凋亡過程中的功能與作用進(jìn)行了研究,主要結(jié)果如下:1.應(yīng)用免疫組織化學(xué)染色、qRT-PCR和Western blotting方法,研究了ATF6在雌性小鼠發(fā)情周期不同階段的卵巢、輸卵管以及子宮中的表達(dá)模式和特點(diǎn)。結(jié)果發(fā)現(xiàn)ATF6的mRNA和蛋白表達(dá)水平在小鼠發(fā)情周期不同階段的卵巢、輸卵管和子宮中呈現(xiàn)一定的規(guī)律性。ATF6蛋白的表達(dá)主要定位于卵巢的卵母細(xì)胞、顆粒細(xì)胞和黃體細(xì)胞,輸卵管的腔上皮細(xì)胞和子宮的腔上皮細(xì)胞與腺上皮細(xì)胞中。在卵巢、輸卵管和子宮中,ATF6的mRNA和蛋白表達(dá)水平在發(fā)情前期與發(fā)情期處于相對(duì)較低的水平,從發(fā)情后期至間情期逐漸升高,提示ATF6可能參與了雌性小鼠的生殖過程,ATF6的表達(dá)可能受到卵巢中P4和E2的影響。2.研究了ATF6在小鼠早期正常妊娠、假孕、延遲著床與著床激活、人工誘導(dǎo)子宮蛻膜化、類固醇激素誘導(dǎo)與拮抗劑處理等子宮中的表達(dá)與定位特點(diǎn)。結(jié)果發(fā)現(xiàn)ATF6的mRNA和蛋白表達(dá)水平在正常妊娠第5 d植入“窗口期”的子宮中顯著升高,ATF6蛋白在植入位點(diǎn)附近的腔上皮和基質(zhì)細(xì)胞中呈現(xiàn)高表達(dá);ATF6在假孕第5 d子宮中的表達(dá)量顯著降低;ATF6的mRNA和蛋白在著床激活模型子宮中的表達(dá)量顯著高于對(duì)照組,ATF6蛋白在著床激活模型子宮中植入位點(diǎn)附近的基質(zhì)細(xì)胞、腔上皮細(xì)胞中呈現(xiàn)高表達(dá),推測(cè)ATF6可能參與了早期胚胎的黏附與植入過程,胚胎的存在可能影響ATF6的表達(dá)。ATF6的mRNA和蛋白在正常妊娠第6~8 d子宮中的表達(dá)水平逐漸上升,在妊娠第6 d的初級(jí)蛻膜區(qū)(PDZ)中檢測(cè)到了ATF6蛋白,ATF6高表達(dá)于妊娠7~8 d的次級(jí)蛻膜區(qū)(sdz);并且在人工誘導(dǎo)蛻膜化模型子宮中,atf6的表達(dá)量顯著高于對(duì)照組,推測(cè)atf6可能參與了早期妊娠子宮中基質(zhì)細(xì)胞的蛻膜化及蛻膜區(qū)的形成過程。在類固醇激素與相應(yīng)拮抗劑處理模型子宮中的腔上皮、腺上皮細(xì)胞中檢測(cè)到了atf6蛋白的表達(dá)。與對(duì)照組即未經(jīng)處理的子宮相比,atf6的mrna和蛋白表達(dá)水平發(fā)生了顯著變化,推測(cè)atf6在子宮中的表達(dá)受到p4和e2的共同調(diào)節(jié)。3.應(yīng)用慢病毒載體介導(dǎo)的shrna干擾技術(shù),成功構(gòu)建了針對(duì)小鼠atf6基因的shrna干擾重組慢病毒載體,并對(duì)其干擾效率進(jìn)行了相關(guān)檢測(cè)與驗(yàn)證。結(jié)果顯示包裝獲得的atf6基因重組干擾慢病毒的滴度約為7×107tu/ml,對(duì)小鼠raw264.7巨噬細(xì)胞的感染效率達(dá)到90%以上。同時(shí),篩選出了能夠有效抑制atf6基因表達(dá)的慢病毒干擾載體,其在小鼠raw264.7巨噬細(xì)胞中的干擾效率在80%左右,可以滿足對(duì)atf6基因生物學(xué)功能進(jìn)行實(shí)驗(yàn)研究的要求。4.研究了atf6在性成熟小鼠不同發(fā)育階段的卵泡和體外原代培養(yǎng)的顆粒細(xì)胞中的表達(dá)情況。結(jié)果發(fā)現(xiàn)atf6廣泛表達(dá)于性成熟小鼠卵泡不同生長發(fā)育時(shí)期的卵母細(xì)胞和顆粒細(xì)胞中,atf6在顆粒細(xì)胞中的蛋白表達(dá)水平受到fsh/lh的調(diào)節(jié)。應(yīng)用針對(duì)小鼠atf6基因的干擾慢病毒感染了體外原代培養(yǎng)的小鼠顆粒細(xì)胞,發(fā)現(xiàn)shatf6干擾慢病毒能夠有效抑制atf6基因在顆粒細(xì)胞中的表達(dá),干擾效率在80%左右。應(yīng)用qrt-pcr、westernblotting、流式細(xì)胞術(shù)和elisa等方法,對(duì)抑制了atf6基因表達(dá)的顆粒細(xì)胞進(jìn)行檢測(cè)后發(fā)現(xiàn),抑制atf6基因的表達(dá)能夠通過下調(diào)p53、caspase-3、chop和上調(diào)bcl-2與grp78等凋亡調(diào)控關(guān)鍵基因的表達(dá)水平而抑制顆粒細(xì)胞的凋亡;能夠通過抑制cyclina1、cyclinb1和cyclind2等細(xì)胞周期調(diào)控關(guān)鍵基因的表達(dá)干擾顆粒細(xì)胞的正常細(xì)胞周期分布;并且能夠通過上調(diào)cyp11a1、star、cyp19a1和下調(diào)cyp1b1等類固醇激素分泌調(diào)控關(guān)鍵基因的表達(dá),而促進(jìn)孕酮和雌激素在顆粒細(xì)胞中的分泌;還能夠?qū)εc卵泡生長發(fā)育相關(guān)的功能性基因has2、ptgs2、ptgfr和igfbp4等在顆粒細(xì)胞中的表達(dá)產(chǎn)生顯著影響。5.應(yīng)用cck-8法和westernblotting研究了赤霉烯酮對(duì)小鼠顆粒細(xì)胞的細(xì)胞毒性作用,發(fā)現(xiàn)赤霉烯酮能夠顯著抑制小鼠顆粒細(xì)胞的活力,呈現(xiàn)出一定的濃度與時(shí)間依賴性的特點(diǎn),并且赤霉烯酮能夠影響atf6在顆粒細(xì)胞中的表達(dá)。應(yīng)用qrt-pcr、westernblotting、流式細(xì)胞術(shù)和elisa等方法,研究了atf6在赤霉烯酮誘導(dǎo)小鼠顆粒細(xì)胞凋亡過程中的作用,發(fā)現(xiàn)抑制atf6的表達(dá)能夠通過下調(diào)p53、caspase-3、bax和上調(diào)bcl-2等凋亡調(diào)控關(guān)鍵基因的表達(dá)而抑制赤霉烯酮誘導(dǎo)的顆粒細(xì)胞凋亡;能夠通過上調(diào)hsd3b2和cyp19a1等類固醇激素合成調(diào)控關(guān)鍵基因的表達(dá)而緩解赤霉烯酮誘導(dǎo)的顆粒細(xì)胞中p4和e2的分泌功能障礙;但抑制atf6對(duì)赤霉烯酮引起的顆粒細(xì)胞周期阻滯無顯著影響。在赤霉烯酮誘導(dǎo)小鼠顆粒細(xì)胞凋亡的過程中,赤霉烯酮能夠激活內(nèi)質(zhì)網(wǎng)應(yīng)激,促進(jìn)atf6、grp78和chop等內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白在顆粒細(xì)胞中的表達(dá);抑制atf6能夠通過上調(diào)GRP78和下調(diào)CHOP的表達(dá)水平而降低細(xì)胞的凋亡率;并且赤霉烯酮還能夠激活細(xì)胞的自噬,抑制ATF6可能通過增強(qiáng)細(xì)胞的自噬而對(duì)細(xì)胞起到一定的保護(hù)作用。以上結(jié)果表明,ATF6在雌性小鼠的胚胎植入、子宮蛻膜化以及卵巢顆粒細(xì)胞的凋亡等主要生殖過程中發(fā)揮了重要作用,為進(jìn)一步深入研究ATF6的生物學(xué)功能和內(nèi)質(zhì)網(wǎng)應(yīng)激在哺乳動(dòng)物生殖過程中的作用提供了研究基礎(chǔ)和理論依據(jù)。
[Abstract]:Activating transcription factor 6 (ATF6) is located in the endoplasmic reticulum type II transmembrane protein belonging to the CREB family transcription factor.ATF6 as a major baroreceptor endoplasmic reticulum stress signaling pathway in (Stress Sensor) protein, endoplasmic reticulum stress, it can in many tissues and endoplasmic reticulum stress related the response element regulated gene co synthesis and degradation of.ATF6 cell protein is widely expressed in the body, organs and cells, such as placenta, uterine decidual tissue, liver, heart, fetal fibroblast cells and RAW264.7 macrophages. The existing research shows that endoplasmic reticulum stress and its signal transduction pathway related genes in early embryos implantation of mammalian, decidual endometrial stromal cells and ovarian function to maintain and play a series of reproductive processes play important regulation for Use. Therefore, further reveal the role of ATF6 and its mediated endoplasmic reticulum stress signaling pathway in the reproductive process of female mammals, for a comprehensive understanding of the molecular mechanism of mammalian reproductive regulation, has important value to explore the new target for the regulation of animal breeding. In this study, a mouse model of immunohistochemical staining, qRT-PCR, Western blotting, shRNA interference, flow cytometry and TUNEL technology and method of network function and role stress related gene ATF6 in uterine and ovarian granulosa cell apoptosis of mouse embryo implantation in the period studied, the main results are as follows: 1. immunohistochemical staining, qRT-PCR and Western blotting, of ATF6 in the different stages of the estrous cycle of female mice ovary, expression pattern and characteristics of the fallopian tube and uterus. The results showed that mRNA ATF6 and protein expression of water In different stages of estrous cycle of the ovary, the main expression showed a certain pattern of.ATF6 protein in the oviduct and uterus in ovarian oocytes, granulosa cells and luteal cells, epithelial cells and uterine oviduct luminal epithelium and glandular epithelial cells. In ovary, oviduct and uterus in the mRNA and protein expression levels of ATF6 at a relatively low level in proestrus and estrus, gradually increased from metestrous to diestrus, suggesting that ATF6 may be involved in the reproductive process of female mice, the expression of ATF6 may be affected by P4 and E2 in the ovary of.2. ATF6 was studied in normal pregnancy. In early pseudopregnancy, delayed implantation and implantation of artificial induced activation, uterine decidualization, expression and localization of the steroid hormone induction and antagonist treatment in the womb. The results showed that the expression of mRNA protein and ATF6 of water Ping Zaizheng Often pregnancy fifth d into the "window period" of the uterus increased significantly, ATF6 protein showed high expression in the luminal epithelium and stromal cells in the vicinity of the implantation site; the expression of ATF6 in fifth D in the amount of pseudopregnant uterus decreased significantly; mRNA and protein ATF6 activation model in the womb in implantation was significantly higher than that of control group ATF6 protein, activation model of uterine stromal cells implanted at the implantation site near the cavity, showed high expression in epithelial cells, suggesting that ATF6 may be involved in the adhesion and implantation process of embryos, embryos may influence the expression level of mRNA and protein expression of.ATF6 ATF6 in the 6~8 of D in normal pregnancy in the uterus increased gradually in the primary decidual zone pregnancy sixth D (PDZ) was detected in the ATF6 protein, the secondary decidual zone with high expression of ATF6 in pregnancy 7~8 D (SDZ); and in the artificial decidualization of uterine model, the expression of ATF6 significantly That is higher than the control group, the formation process of ATF6 may be involved in early pregnancy in uterine stromal cell decidualization and decidual zone. In steroid hormone antagonists and the corresponding processing model in uterine luminal epithelial and glandular epithelial cells were detected in the expression of ATF6 protein. And the control group compared to untreated uterus, occurrence a significant change in the expression level of mRNA ATF6 and protein expression, suggesting that ATF6 in the uterus was regulated by shRNA interference.3. by lentiviral vector mediated P4 and E2, successfully constructed shRNA interference recombinant lentiviral vector targeting mouse ATF6 gene, and the interference efficiency were tested and verified. Results showed that the titers of ATF6 recombinant lentivirus packaging obtained is about 7 * 107tu/ml, transfection efficiency in RAW264.7 cells reached more than 90%. At the same time, selected can effectively restrain a The expression of TF6 gene lentivirus interference vector in mouse RAW264.7 macrophages in the interference efficiency was about 80%, the expression can be studied by ATF6 in mature mouse follicles in various stages of development and in vitro primary cultured granulosa cells have carried out the experimental study on the biological functions of ATF6 gene. The results meet the requirements of.4. ATF6 wide the expression of oocyte and granulosa cells in mature mouse follicles at different growth stages, the expression of ATF6 in granulosa cells regulated by fsh/lh protein levels. Application of targeting mouse ATF6 gene lentivirus infected mouse granulosa cells cultured in vitro, found the expression of shatf6 interference lentivirus can effectively inhibit ATF6 gene in granulosa cells, the interference efficiency was about 80%. The application of qRT-PCR, westernblotting, flow cytometry and ELISA method, the inhibition of ATF6 gene The expression of granulosa cells were detected after the discovery, inhibit the expression of ATF6 gene can downregulate p53, Caspase-3, chop expression and up regulation of Bcl-2 and GRP78 and apoptosis regulation of key genes and inhibit the apoptosis of granulosa cells; can inhibit cyclina1, normal expression of cell cycle disturbance cyclinB1 granule cells and cyclind2 cell cycle regulation of key genes the distribution; and can through the upregulation of CYP11A1, star, CYP19A1 and CYP1B1 down-regulation of steroid hormone secretion to regulate the expression of key genes, and promotes progesterone and estrogen secretion in granulosa cells; also can PTGS2 on the growth and development of functional gene Has2 associated with follicular, significantly affect.5. and westernblotting studied by CCK-8 method cytotoxic effects of zearalenone on mouse granulosa cells the expression of ptgfr and IGFBP4 in granulosa cells, zearalenone can be found Could significantly inhibit murine granulosa cell viability, characterized by a concentration and time dependent, and zearalenone can affect the expression of ATF6 in granulosa cells. The application of qRT-PCR, westernblotting, flow cytometry and ELISA method, studied the ATF6 induced apoptosis of mouse granulosa cells in red mycophenolate ketene, found that inhibiting the expression of ATF6 can downregulate p53, Caspase-3, Bax and Bcl-2 up-regulated expression of key genes and inhibiting apoptosis of granulosa cell apoptosis induced by zearalenone; can regulate the expression of up regulation of HSD3B2 and CYP19A1 and other steroid hormone biosynthesis key genes P4 and E2 in granulosa cells and relieve gibberellenic ketone induced the secretion dysfunction; but the inhibition of granule cell cycle arrest ATF6 on zearalenone caused no significant effect. The process of induced apoptosis of mouse granulosa cells in zearalenone in , zearalenone can activate the endoplasmic reticulum stress, promote the expression of ATF6, GRP78 and chop of endoplasmic reticulum stress related proteins in granulosa cells; inhibition of ATF6 through upregulation of GRP78 and downregulation of CHOP expression and decreased the apoptosis rate; and zearalenone can also activate autophagy, may inhibit ATF6 by enhancing the autophagy and protective effects on cells. These results indicate that ATF6 in female mouse embryo implantation, played an important role in uterine decidualization and ovarian granulosa cell apoptosis in the main reproductive process, provides the research foundation and theoretical basis for further study of biological function of ATF6 and ER net stress in mammalian reproduction.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S814
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本文編號(hào)：1537481