ATF6在小鼠胚胎植入期子宫和卵巢颗粒细胞凋亡过程中的作用研究

发布时间：2018-02-26 09:39

  本文关键词： 雌性小鼠 胚胎植入 蜕膜化 类固醇激素 颗粒细胞　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

【摘要】：Activating transcription factor 6(ATF6)是一个位于细胞内质网膜上的II型跨膜蛋白,属于CREB转录因子家族成员。ATF6作为内质网应激信号转导通路中的一个主要压力感受器(Stress Sensor)蛋白,发生内质网应激时,它能够与相关的内质网应激响应基因元件协同而调节细胞中蛋白质的合成与降解。ATF6广泛表达于机体的多种组织、器官和细胞中,如胎盘、子宫的蜕膜组织、肝脏、心脏、胎儿成纤维细胞和RAW264.7巨噬细胞等。现有研究表明,内质网应激及其信号转导通路中的相关基因在哺乳动物的早期胚胎植入、子宫内膜基质细胞蜕膜化和卵巢功能的维持与发挥等一系列生殖过程中发挥了重要的调节作用。因而,进一步深入揭示ATF6及其介导的内质网应激信号通路在雌性哺乳动物生殖过程中的作用,对于全面认识哺乳动物生殖调控的分子机制、探寻调控动物繁殖的新靶点具有重要价值。本研究以小鼠为模型,应用免疫组织化学染色、qRT-PCR、Western blotting、shRNA干扰、流式细胞术和TUNEL等技术和方法,对内网应激相关基因ATF6在小鼠胚胎植入时期的子宫和卵巢颗粒细胞凋亡过程中的功能与作用进行了研究,主要结果如下:1.应用免疫组织化学染色、qRT-PCR和Western blotting方法,研究了ATF6在雌性小鼠发情周期不同阶段的卵巢、输卵管以及子宫中的表达模式和特点。结果发现ATF6的mRNA和蛋白表达水平在小鼠发情周期不同阶段的卵巢、输卵管和子宫中呈现一定的规律性。ATF6蛋白的表达主要定位于卵巢的卵母细胞、颗粒细胞和黄体细胞,输卵管的腔上皮细胞和子宫的腔上皮细胞与腺上皮细胞中。在卵巢、输卵管和子宫中,ATF6的mRNA和蛋白表达水平在发情前期与发情期处于相对较低的水平,从发情后期至间情期逐渐升高,提示ATF6可能参与了雌性小鼠的生殖过程,ATF6的表达可能受到卵巢中P4和E2的影响。2.研究了ATF6在小鼠早期正常妊娠、假孕、延迟着床与着床激活、人工诱导子宫蜕膜化、类固醇激素诱导与拮抗剂处理等子宫中的表达与定位特点。结果发现ATF6的mRNA和蛋白表达水平在正常妊娠第5 d植入“窗口期”的子宫中显著升高,ATF6蛋白在植入位点附近的腔上皮和基质细胞中呈现高表达;ATF6在假孕第5 d子宫中的表达量显著降低;ATF6的mRNA和蛋白在着床激活模型子宫中的表达量显著高于对照组,ATF6蛋白在着床激活模型子宫中植入位点附近的基质细胞、腔上皮细胞中呈现高表达,推测ATF6可能参与了早期胚胎的黏附与植入过程,胚胎的存在可能影响ATF6的表达。ATF6的mRNA和蛋白在正常妊娠第6~8 d子宫中的表达水平逐渐上升,在妊娠第6 d的初级蜕膜区(PDZ)中检测到了ATF6蛋白,ATF6高表达于妊娠7~8 d的次级蜕膜区(sdz);并且在人工诱导蜕膜化模型子宫中,atf6的表达量显著高于对照组,推测atf6可能参与了早期妊娠子宫中基质细胞的蜕膜化及蜕膜区的形成过程。在类固醇激素与相应拮抗剂处理模型子宫中的腔上皮、腺上皮细胞中检测到了atf6蛋白的表达。与对照组即未经处理的子宫相比,atf6的mrna和蛋白表达水平发生了显著变化,推测atf6在子宫中的表达受到p4和e2的共同调节。3.应用慢病毒载体介导的shrna干扰技术,成功构建了针对小鼠atf6基因的shrna干扰重组慢病毒载体,并对其干扰效率进行了相关检测与验证。结果显示包装获得的atf6基因重组干扰慢病毒的滴度约为7×107tu/ml,对小鼠raw264.7巨噬细胞的感染效率达到90%以上。同时,筛选出了能够有效抑制atf6基因表达的慢病毒干扰载体,其在小鼠raw264.7巨噬细胞中的干扰效率在80%左右,可以满足对atf6基因生物学功能进行实验研究的要求。4.研究了atf6在性成熟小鼠不同发育阶段的卵泡和体外原代培养的颗粒细胞中的表达情况。结果发现atf6广泛表达于性成熟小鼠卵泡不同生长发育时期的卵母细胞和颗粒细胞中,atf6在颗粒细胞中的蛋白表达水平受到fsh/lh的调节。应用针对小鼠atf6基因的干扰慢病毒感染了体外原代培养的小鼠颗粒细胞,发现shatf6干扰慢病毒能够有效抑制atf6基因在颗粒细胞中的表达,干扰效率在80%左右。应用qrt-pcr、westernblotting、流式细胞术和elisa等方法,对抑制了atf6基因表达的颗粒细胞进行检测后发现,抑制atf6基因的表达能够通过下调p53、caspase-3、chop和上调bcl-2与grp78等凋亡调控关键基因的表达水平而抑制颗粒细胞的凋亡;能够通过抑制cyclina1、cyclinb1和cyclind2等细胞周期调控关键基因的表达干扰颗粒细胞的正常细胞周期分布;并且能够通过上调cyp11a1、star、cyp19a1和下调cyp1b1等类固醇激素分泌调控关键基因的表达,而促进孕酮和雌激素在颗粒细胞中的分泌;还能够对与卵泡生长发育相关的功能性基因has2、ptgs2、ptgfr和igfbp4等在颗粒细胞中的表达产生显著影响。5.应用cck-8法和westernblotting研究了赤霉烯酮对小鼠颗粒细胞的细胞毒性作用,发现赤霉烯酮能够显著抑制小鼠颗粒细胞的活力,呈现出一定的浓度与时间依赖性的特点,并且赤霉烯酮能够影响atf6在颗粒细胞中的表达。应用qrt-pcr、westernblotting、流式细胞术和elisa等方法,研究了atf6在赤霉烯酮诱导小鼠颗粒细胞凋亡过程中的作用,发现抑制atf6的表达能够通过下调p53、caspase-3、bax和上调bcl-2等凋亡调控关键基因的表达而抑制赤霉烯酮诱导的颗粒细胞凋亡;能够通过上调hsd3b2和cyp19a1等类固醇激素合成调控关键基因的表达而缓解赤霉烯酮诱导的颗粒细胞中p4和e2的分泌功能障碍;但抑制atf6对赤霉烯酮引起的颗粒细胞周期阻滞无显著影响。在赤霉烯酮诱导小鼠颗粒细胞凋亡的过程中,赤霉烯酮能够激活内质网应激,促进atf6、grp78和chop等内质网应激相关蛋白在颗粒细胞中的表达;抑制atf6能够通过上调GRP78和下调CHOP的表达水平而降低细胞的凋亡率;并且赤霉烯酮还能够激活细胞的自噬,抑制ATF6可能通过增强细胞的自噬而对细胞起到一定的保护作用。以上结果表明,ATF6在雌性小鼠的胚胎植入、子宫蜕膜化以及卵巢颗粒细胞的凋亡等主要生殖过程中发挥了重要作用,为进一步深入研究ATF6的生物学功能和内质网应激在哺乳动物生殖过程中的作用提供了研究基础和理论依据。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S814
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本文编号：1537481