本文選題：孕激素　切入點(diǎn)：胚胎植入　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景與目的:孕激素(Progesterone,P)在胚胎發(fā)育成熟和著床過(guò)程中起重要作用,孕激素不足可導(dǎo)致胚胎發(fā)育不良,及妊娠終止引起流產(chǎn)。孕激素通過(guò)調(diào)節(jié)與著床有關(guān)分子的表達(dá)促進(jìn)胚胎的增殖和黏附能力。多種病理和生理過(guò)程受到蛋白質(zhì)糖基化的影響,其中包括了胚胎發(fā)育與著床過(guò)程。O-糖基化和N-糖基化是蛋白質(zhì)糖基化的主要形式。蛋白質(zhì)的O-巖藻糖基化受蛋白質(zhì)O-巖藻糖基轉(zhuǎn)移酶(po FUTs,protein-fucosyltransferases)的催化,而po FUTs在促進(jìn)胚胎發(fā)育與著床過(guò)程中的作用未見(jiàn)報(bào)道。激活蛋白-1(AP-1)是一種轉(zhuǎn)錄因子,轉(zhuǎn)錄因子家族由Jun和Fos的細(xì)胞同源物組成,參與細(xì)胞增殖、分化、和侵襲過(guò)程和內(nèi)分泌基因的調(diào)節(jié)。研究發(fā)現(xiàn),AP-1(c-Jun/c-Fos)廣泛表達(dá)于人胎盤(pán)中,活化的AP-1具有調(diào)控胎盤(pán)的形成和胎兒發(fā)育的作用。本研究擬探討孕激素對(duì)蛋白質(zhì)O-巖藻糖基化的調(diào)控作用及影響胚胎增殖和著床過(guò)程。研究結(jié)果表明JAR細(xì)胞經(jīng)孕激素作用,增加AP-1(c-Jun/c-Fos)的磷酸化表達(dá),上調(diào)poFUT1的基因表達(dá),并促進(jìn)胚胎細(xì)胞的黏附能力與增殖能力。實(shí)驗(yàn)有助于揭示巖藻糖在著床糖生殖生物學(xué)中的作用及其機(jī)制,為臨床醫(yī)學(xué)提供研究及診斷治療的新思路。方法:1.利用ELISA和Western blot檢測(cè)健康未孕、早孕和流產(chǎn)婦女血清中孕激素和poFUT1的蛋白表達(dá)水平。通過(guò)免疫熒光的方法驗(yàn)證人絨毛組織中poFUT1的定位和表達(dá)。2.利用Western blot,免疫熒光和Real-time PCR等方法,觀察孕激素對(duì)胚胎滋養(yǎng)層細(xì)胞中poFUT1基因和蛋白表達(dá)水平的影響,以及孕激素與米非司酮聯(lián)合應(yīng)用對(duì)poFUT1基因和蛋白表達(dá)水平的影響。3.利用Western blot,免疫熒光和Real-time PCR等方法,觀察孕激素影響poFUT1的表達(dá)及對(duì)細(xì)胞增殖黏附功能的改變。4.為了探討孕激素是如何調(diào)控poFUT1表達(dá),我們采用Western blot、EMSA和免疫熒光方法確定孕激素可激活轉(zhuǎn)錄因子AP-1的轉(zhuǎn)錄活性。5.利用染色質(zhì)免疫沉淀技術(shù)檢測(cè)轉(zhuǎn)錄因子AP-1直接作用在poFUT1的啟動(dòng)子區(qū)域,促進(jìn)poFUT1轉(zhuǎn)錄,并且通過(guò)Western blot和Real-time PCR檢測(cè)瞬時(shí)轉(zhuǎn)染c-Jun/c-Fos siRNA干擾質(zhì)粒和AP-1抑制劑(TIIA)處理后poFUT1基因和蛋白表達(dá)的變化。6.體外著床模型是在熒光顯微鏡下,觀察和統(tǒng)計(jì)黏附率,檢測(cè)了包括正常組、孕激素組、孕激素聯(lián)合米非司酮組、轉(zhuǎn)染c-Jun/c-Fos siRNA干擾質(zhì)粒組,AP-1抑制劑(TIIA)組以及分別聯(lián)合孕激素等9組的胚胎黏附率。并且利用Western blot、免疫熒光和CCK-8檢測(cè)JAR細(xì)胞增殖能力。結(jié)果:1.收集臨床樣品包括健康未孕、正常早孕和先兆流產(chǎn)女性血清進(jìn)行ELISA檢測(cè)發(fā)現(xiàn):正常早孕婦女血清中,孕激素和poFUT1的含量均高于先兆流產(chǎn)患者和健康未孕的女性;而先兆流產(chǎn)患者血清中,孕激素和poFUT1的含量比早孕婦女低。孕激素和poFUT1在血清中的表達(dá)變化呈正相關(guān)。通過(guò)免疫熒光確定人早孕絨毛中poFUT1的定位和表達(dá)。2.孕激素促進(jìn)poFUT1的基因和蛋白水平的表達(dá)。經(jīng)過(guò)不同濃度的孕激素和不同作用時(shí)間處理后,通過(guò)Real-time PCR和Western blot等方法檢測(cè)發(fā)現(xiàn),孕激素能顯著促進(jìn)JAR細(xì)胞poFUT1基因和蛋白的表達(dá);Real-time PCR、Western blot和免疫熒光結(jié)果表明,聯(lián)合應(yīng)用米非司酮(RU486)可抑制孕激素的效用;瞬時(shí)轉(zhuǎn)染poFUT1 siRNA后,poFUT1表達(dá)明顯降低,聯(lián)合孕激素處理后可回調(diào)poFUT1的表達(dá)。3.孕激素促進(jìn)poFUT1的表達(dá)并調(diào)節(jié)JAR細(xì)胞的增殖與黏附的能力。Real-time PCR、Western blot和免疫熒光結(jié)果發(fā)現(xiàn),干擾poFUT1的JAR細(xì)胞中合并應(yīng)用孕激素可以恢復(fù)poFUT1的表達(dá)。通過(guò)Western blot、CCK-8和免疫熒光等方法分析發(fā)現(xiàn)孕激素調(diào)控poFUT1的表達(dá)影響JAR細(xì)胞的增殖和黏附能力。4.孕激素激活轉(zhuǎn)錄因子AP-1的轉(zhuǎn)錄活性。Western blot、EMSA和免疫熒光結(jié)果發(fā)現(xiàn),孕激素(10-5mol/L)可上調(diào)轉(zhuǎn)錄因子AP-1(c-Jun/c-Fos)的表達(dá)水平,并促進(jìn)其在核中的積累。5.AP-1直接影響靶基因poFUT1的轉(zhuǎn)錄,Real-time PCR、Western blot結(jié)果顯示,與對(duì)照組相比,抑制了AP-1的轉(zhuǎn)錄激活能力可降低poFUT1的表達(dá)水平。通過(guò)CHIP實(shí)驗(yàn)進(jìn)一步確定了AP-1直接作用在poFUT1啟動(dòng)子區(qū)域,引起下游靶基因poFUT1轉(zhuǎn)錄并調(diào)控其表達(dá)變化。6.孕激素通過(guò)激活轉(zhuǎn)錄因子AP-1上調(diào)poFUT1表達(dá)水平。孕激素可調(diào)控poFUT1的表達(dá),poFUT1的表達(dá)變化可影響胚胎細(xì)胞增殖能力。通過(guò)Western blot、CCK-8和免疫熒光分析發(fā)現(xiàn),與對(duì)照組相比,孕激素能提高JAR細(xì)胞的增殖能力;c-Jun/c-Fos siRNA能降低JAR細(xì)胞的增殖能力;聯(lián)合孕激素處理可一定程度的回調(diào)由c-Jun/c-Fos siRNA引起JAR細(xì)胞增殖能力降低的現(xiàn)象;同時(shí),孕激素也能夠回調(diào)由AP-1抑制劑(TIIA)引起JAR細(xì)胞低的增殖能力;研究發(fā)現(xiàn),孕激素可提高胚胎細(xì)胞的增殖能力。7.利用體外黏附模型,觀察胚胎細(xì)胞的體外黏附率。孕激素可提高JAR細(xì)胞的黏附率;瞬時(shí)轉(zhuǎn)染c-Jun/c-Fos siRNA干擾質(zhì)粒可降低JAR細(xì)胞的黏附率;同時(shí),孕激素能夠恢復(fù)c-Jun/c-Fos siRNA預(yù)處理的JAR細(xì)胞的低黏附率;孕激素也能夠恢復(fù)預(yù)處理AP-1抑制劑TIIA的JAR細(xì)胞低黏附率;進(jìn)一步的研究發(fā)現(xiàn)孕激素可恢復(fù)poFUT1 siRNA導(dǎo)致的胚胎較低的黏附率。孕激素通過(guò)促進(jìn)poFUT1的表達(dá)提高了胚胎的黏附能力。結(jié)論:1.正常早孕時(shí)期女性血清中孕激素和poFUT1的含量較先兆流產(chǎn)患者高,且呈正相關(guān)性。人絨毛組織上有poFUT1的表達(dá)。2.孕激素促進(jìn)poFUT1基因與蛋白的表達(dá)水平。3.孕激素激活轉(zhuǎn)錄因子AP-1(c-Jun/c-Fos),并上調(diào)poFUT1的表達(dá)。4.孕激素促進(jìn)胚胎細(xì)胞增殖能力。5.孕激素通過(guò)激活轉(zhuǎn)錄因子AP-1(c-Jun/c-Fos)的轉(zhuǎn)錄活性,進(jìn)而調(diào)控靶基因poFUT1的轉(zhuǎn)錄,促進(jìn)胚胎細(xì)胞與子宮內(nèi)膜細(xì)胞的黏附。
[Abstract]:Background and purpose: progesterone (Progesterone, P) play an important role in the maturation of embryo implantation and process, progesterone deficiency can lead to embryonic dysplasia, and progesterone induced termination of pregnancy abortion. The ability to promote the proliferation and adhesion of embryo implantation and by regulating the expression of related molecules. A variety of physiological and pathological processes affected by protein glycosylation, including the process of embryo development and implantation of.O- glycosylation and N- glycosylation is the main form of glycosylated proteins. O- fucosylated proteins by protein O- fucosyltransferase (PO FUTs, protein-fucosyltransferases) and Po FUTs in catalysis, promote the process of embryo development and implantation. The role has not been reported. Activated protein -1 (AP-1) is a transcription factor family of transcription factors by Jun and Fos cellular homologues, involved in cell proliferation, differentiation, and invasion process and Regulating the secretion of genes. The study found that AP-1 (c-Jun/c-Fos) is widely expressed in human placenta, the activation of AP-1 can regulate the formation of the placenta and fetal development. This study intends to explore the role of progesterone on protein O- fucosylated regulation and proliferation and embryo implantation process. The results show that the JAR cells were treated with progestin. AP-1 (c-Jun/c-Fos), increased the expression of phosphorylation, upregulating the expression of poFUT1 gene, and to promote the adhesion and proliferation ability of embryonic cells. The results are helpful to reveal the fucose sugar in implantation in the reproductive biology effect and mechanism, and provides a new way to research the diagnosis and treatment for clinical medicine. Methods: using ELISA and 1. Western blot detection of the expression level of healthy non pregnant, pregnancy and abortion protein progesterone and poFUT1 in serum. The localization of poFUT1 by immunofluorescence test in human chorionic tissues And the expression of.2. by Western blot, immunofluorescence and Real-time PCR method to observe the effect of progesterone on the expression level of poFUT1 gene and protein in embryonic trophoblast cells, and the combined use of progesterone and mifepristone on poFUT1 gene and protein expression and the influence of.3. levels by Western blot, immunofluorescence and Real-time PCR methods, observe the effects of progesterone on poFUT1 the expression and change of.4. on cell proliferation adhesion function in order to investigate the progesterone is how to regulate the expression of poFUT1, we use Western blot EMSA and immunofluorescence method to determine the transcriptional activity of.5. progesterone can activate transcription factor AP-1 by chromatin immunoprecipitation assays of AP-1 transcription factor technology directly precipitated in the poFUT1 promoter region, promote poFUT1 transcription and through the Western blot and Real-time PCR detection of transient transfection of c-Jun/c-Fos siRNA plasmid and AP-1 interference suppression Preparation of (TIIA) changes of.6. implantation model in vitro poFUT1 gene and protein expression after treatment is under the fluorescence microscope, observation and statistics of the rate of adhesion was detected including normal group, progesterone group, progesterone and mifepristone group, transfection of c-Jun/c-Fos siRNA plasmid group, AP-1 inhibitor (TIIA) group and 9 group were combined with progesterone the adhesion rate of embryo and the use of Western. Blot, immunofluorescence and CCK-8 JAR detection ability of cell proliferation. Results: 1. collected clinical samples including healthy non pregnant women, normal pregnancy and threatened abortion of ELISA in serum were detected: the serum of normal pregnancy, progesterone and poFUT1 content were higher than that of threatened abortion patients and healthy non pregnant women the serum of patients with threatened abortion; however, the content of progesterone and poFUT1 than pregnant women. Low expression of progesterone and poFUT1 in serum were positively correlated with immune. Fluorescence to determine the position of poFUT1 in the villi and the expression of.2. and progesterone promotes the expression of poFUT1 gene and protein level. After different concentrations of progesterone and different time after treatment, detected by Real-time PCR and Western blot found that progesterone can significantly promote the expression of poFUT1 gene and protein in JAR cells; Real-time PCR, Western blot and immunofluorescence results show that the combined application of mifepristone (RU486) can inhibit the progesterone effect; transient transfection of poFUT1 siRNA, poFUT1 expression was significantly reduced after the treatment of combined progestin adjusting the expression of poFUT1.3. and progesterone can promote the expression of poFUT1 and JAR cell proliferation and adhesion regulating ability of.Real-time PCR, Western blot and immunofluorescence. The results showed that interfering the expression of poFUT1 JAR cells in combination with progesterone can restore poFUT1. By Western blot, CCK-8 and immune Fluorescence analysis found that progesterone regulates poFUT1 expression JAR cell proliferation and adhesion of.4. progesterone activated transcription factor AP-1 transcription activity of.Western blot EMSA, and immunofluorescence results showed that progesterone (10-5mol/L) can upregulate the transcription factor AP-1 (c-Jun/ c-Fos) expression levels, and promote its nuclear accumulation in.5.AP-1 effect of transcription of the target gene of poFUT1 Real-time PCR and Western blot results showed that compared with the control group, the inhibition of AP-1 transcriptional activation ability can reduce the expression level of poFUT1. A direct role for AP-1 in the promoter region of poFUT1 was determined by CHIP experiment further, causing downstream target gene poFUT1 transcription and regulation of the expression of.6. and progesterone through the activation of the transcription factor AP-1 up-regulated the expression level of poFUT1. Progesterone can regulate the expression of poFUT1, poFUT1 expression changes can affect embryonic cell proliferation. By Western blot, CCK-8 and immunofluorescence analysis showed that compared with the control group, progesterone can enhance the proliferation of JAR cells; c-Jun/c-Fos siRNA can reduce the proliferation of JAR cells in combination with progesterone treatment; callback to some extent decreases due to the proliferation ability of JAR cells by c-Jun/c-Fos siRNA phenomenon; at the same time, progesterone can also callback by AP-1 inhibitor (TIIA) JAR cells induced by low proliferation; the study found that progesterone can improve the adhesion in vitro model of embryonic.7. cells proliferation, embryonic cells in vitro. The adhesion rate of pregnant hormone can improve the adhesion rate of JAR cells; transient transfection of c-Jun/c-Fos siRNA plasmid can reduce the adhesion rate of JAR cells; meanwhile, progesterone can recovery of c-Jun/c-Fos siRNA pretreatment of JAR cells with low adhesion rate; progesterone can recover pretreatment of AP-1 inhibitor of TIIA JAR cells with low adhesion A further study found that the adhesion rate; progesterone can restore the poFUT1 siRNA leads to embryonic lower rate. Progesterone by promoting the expression of poFUT1 increased the adhesion ability of embryos. Conclusion: the content of progesterone and poFUT1 in serum of 1. normal pregnant women during the period of a threatened abortion were high, and there was a positive correlation between. Human chorionic tissues on poFUT1 the expression of.2. and progesterone promote poFUT1 gene and protein expression levels of.3. and progesterone activates the transcription factor AP-1 (c-Jun/c-Fos), and the increased expression of poFUT1.4. and progesterone promote embryo.5. cell proliferation ability of progesterone through activation of the transcription factor AP-1 (c-Jun/c-Fos) transcription activity, transcription and regulation of target gene poFUT1, promote adhesion of embryonic cells and endometrial cells.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R714

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 王海濱;;妊娠建立和維持的分子機(jī)制[J];中國(guó)基礎(chǔ)科學(xué);2015年05期

2 李軍;杜鑫;Hosseini Moghaddam S.H.;陳玉銀;;蛋白質(zhì)糖基化修飾研究進(jìn)展[J];科技通報(bào);2009年06期

3 陳子江,石玉華;現(xiàn)代不孕癥的診斷治療及研究進(jìn)展[J];現(xiàn)代婦產(chǎn)科進(jìn)展;2004年01期

，

本文編號(hào)：1572463