發(fā)布時間：2018-03-06 00:05

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

【摘要】：背景與目的:孕激素(Progesterone,P)在胚胎發(fā)育成熟和著床過程中起重要作用,孕激素不足可導致胚胎發(fā)育不良,及妊娠終止引起流產(chǎn)。孕激素通過調節(jié)與著床有關分子的表達促進胚胎的增殖和黏附能力。多種病理和生理過程受到蛋白質糖基化的影響,其中包括了胚胎發(fā)育與著床過程。O-糖基化和N-糖基化是蛋白質糖基化的主要形式。蛋白質的O-巖藻糖基化受蛋白質O-巖藻糖基轉移酶(po FUTs,protein-fucosyltransferases)的催化,而po FUTs在促進胚胎發(fā)育與著床過程中的作用未見報道。激活蛋白-1(AP-1)是一種轉錄因子,轉錄因子家族由Jun和Fos的細胞同源物組成,參與細胞增殖、分化、和侵襲過程和內分泌基因的調節(jié)。研究發(fā)現(xiàn),AP-1(c-Jun/c-Fos)廣泛表達于人胎盤中,活化的AP-1具有調控胎盤的形成和胎兒發(fā)育的作用。本研究擬探討孕激素對蛋白質O-巖藻糖基化的調控作用及影響胚胎增殖和著床過程。研究結果表明JAR細胞經(jīng)孕激素作用,增加AP-1(c-Jun/c-Fos)的磷酸化表達,上調poFUT1的基因表達,并促進胚胎細胞的黏附能力與增殖能力。實驗有助于揭示巖藻糖在著床糖生殖生物學中的作用及其機制,為臨床醫(yī)學提供研究及診斷治療的新思路。方法:1.利用ELISA和Western blot檢測健康未孕、早孕和流產(chǎn)婦女血清中孕激素和poFUT1的蛋白表達水平。通過免疫熒光的方法驗證人絨毛組織中poFUT1的定位和表達。2.利用Western blot,免疫熒光和Real-time PCR等方法,觀察孕激素對胚胎滋養(yǎng)層細胞中poFUT1基因和蛋白表達水平的影響,以及孕激素與米非司酮聯(lián)合應用對poFUT1基因和蛋白表達水平的影響。3.利用Western blot,免疫熒光和Real-time PCR等方法,觀察孕激素影響poFUT1的表達及對細胞增殖黏附功能的改變。4.為了探討孕激素是如何調控poFUT1表達,我們采用Western blot、EMSA和免疫熒光方法確定孕激素可激活轉錄因子AP-1的轉錄活性。5.利用染色質免疫沉淀技術檢測轉錄因子AP-1直接作用在poFUT1的啟動子區(qū)域,促進poFUT1轉錄,并且通過Western blot和Real-time PCR檢測瞬時轉染c-Jun/c-Fos siRNA干擾質粒和AP-1抑制劑(TIIA)處理后poFUT1基因和蛋白表達的變化。6.體外著床模型是在熒光顯微鏡下,觀察和統(tǒng)計黏附率,檢測了包括正常組、孕激素組、孕激素聯(lián)合米非司酮組、轉染c-Jun/c-Fos siRNA干擾質粒組,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JAR細胞增殖能力。結果:1.收集臨床樣品包括健康未孕、正常早孕和先兆流產(chǎn)女性血清進行ELISA檢測發(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在血清中的表達變化呈正相關。通過免疫熒光確定人早孕絨毛中poFUT1的定位和表達。2.孕激素促進poFUT1的基因和蛋白水平的表達。經(jīng)過不同濃度的孕激素和不同作用時間處理后,通過Real-time PCR和Western blot等方法檢測發(fā)現(xiàn),孕激素能顯著促進JAR細胞poFUT1基因和蛋白的表達;Real-time PCR、Western blot和免疫熒光結果表明,聯(lián)合應用米非司酮(RU486)可抑制孕激素的效用;瞬時轉染poFUT1 siRNA后,poFUT1表達明顯降低,聯(lián)合孕激素處理后可回調poFUT1的表達。3.孕激素促進poFUT1的表達并調節(jié)JAR細胞的增殖與黏附的能力。Real-time PCR、Western blot和免疫熒光結果發(fā)現(xiàn),干擾poFUT1的JAR細胞中合并應用孕激素可以恢復poFUT1的表達。通過Western blot、CCK-8和免疫熒光等方法分析發(fā)現(xiàn)孕激素調控poFUT1的表達影響JAR細胞的增殖和黏附能力。4.孕激素激活轉錄因子AP-1的轉錄活性。Western blot、EMSA和免疫熒光結果發(fā)現(xiàn),孕激素(10-5mol/L)可上調轉錄因子AP-1(c-Jun/c-Fos)的表達水平,并促進其在核中的積累。5.AP-1直接影響靶基因poFUT1的轉錄,Real-time PCR、Western blot結果顯示,與對照組相比,抑制了AP-1的轉錄激活能力可降低poFUT1的表達水平。通過CHIP實驗進一步確定了AP-1直接作用在poFUT1啟動子區(qū)域,引起下游靶基因poFUT1轉錄并調控其表達變化。6.孕激素通過激活轉錄因子AP-1上調poFUT1表達水平。孕激素可調控poFUT1的表達,poFUT1的表達變化可影響胚胎細胞增殖能力。通過Western blot、CCK-8和免疫熒光分析發(fā)現(xiàn),與對照組相比,孕激素能提高JAR細胞的增殖能力;c-Jun/c-Fos siRNA能降低JAR細胞的增殖能力;聯(lián)合孕激素處理可一定程度的回調由c-Jun/c-Fos siRNA引起JAR細胞增殖能力降低的現(xiàn)象;同時,孕激素也能夠回調由AP-1抑制劑(TIIA)引起JAR細胞低的增殖能力;研究發(fā)現(xiàn),孕激素可提高胚胎細胞的增殖能力。7.利用體外黏附模型,觀察胚胎細胞的體外黏附率。孕激素可提高JAR細胞的黏附率;瞬時轉染c-Jun/c-Fos siRNA干擾質�？山档蚃AR細胞的黏附率;同時,孕激素能夠恢復c-Jun/c-Fos siRNA預處理的JAR細胞的低黏附率;孕激素也能夠恢復預處理AP-1抑制劑TIIA的JAR細胞低黏附率;進一步的研究發(fā)現(xiàn)孕激素可恢復poFUT1 siRNA導致的胚胎較低的黏附率。孕激素通過促進poFUT1的表達提高了胚胎的黏附能力。結論:1.正常早孕時期女性血清中孕激素和poFUT1的含量較先兆流產(chǎn)患者高,且呈正相關性。人絨毛組織上有poFUT1的表達。2.孕激素促進poFUT1基因與蛋白的表達水平。3.孕激素激活轉錄因子AP-1(c-Jun/c-Fos),并上調poFUT1的表達。4.孕激素促進胚胎細胞增殖能力。5.孕激素通過激活轉錄因子AP-1(c-Jun/c-Fos)的轉錄活性,進而調控靶基因poFUT1的轉錄,促進胚胎細胞與子宮內膜細胞的黏附。
[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.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R714

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