皮质醇调控人胎盘合体滋养层细胞芳香化酶表达的分子机制

发布时间：2018-06-29 22:23

本文选题：胎盘 + 合体滋养层细胞　；参考：《复旦大学》2014年博士论文

【摘要】：研究背景根据世界卫生组织提供的数据显示,全球每年约有1500万例早产儿,其中又有约100万例婴儿死于早产并发症,且这个数字仍在逐年上升。然而由于目前人类分娩启动机制尚未完全解析,因此一直缺乏有效的防治早产的手段。于是,解析人类分娩启动机制便成为近年来国际生殖生物学领域的研究热点之一。在大多数哺乳类动物包括人类中,妊娠末期不断增加的雌激素在分娩启动过程中发挥着重要作用。雌激素诱导子宫肌层由静息状态向收缩状态转变,从而为分娩启动做好准备。因此,研究妊娠末期胎盘雌激素合成的调控对阐明分娩启动机制至关重要。之前的研究已经表明,在某些动物如绵羊中,糖皮质激素是分娩启动的关键激素,它能够促进胎盘细胞色素P450c17羟化酶的表达,该酶催化胎盘孕激素向雌激素转化,从而使得糖皮质激素在降低孕激素水平的同时增加雌激素的合成与分泌。然而,灵长类动物包括人类的胎盘缺乏P450c1 7羟化酶,因此无法直接利用孕激素合成雌激素,取而代之的是,人类胎盘可以利用胎儿和母体肾上腺来源的脱氢表雄酮硫酸酯为前体,经过一系列酶促反应尤其是芳香化酶的作用最后生成雌激素。在人类胎盘中,糖皮质激素能否通过影响芳香化酶的表达来促进胎盘雌激素的合成从而参与人类分娩启动过程目前还未见报道。本研究以人胎盘组织及胎盘合体滋养层细胞为研究模型,采用免疫组织化学染色、体外胎盘滋养层细胞培养、实时荧光定量PCR (quantitative real time PCR, qRT-PCR)、蛋白印迹杂交(Western blotting)、酶联免疫测定(Enzyme immunoassay, EIA)、小干扰RNA (small interfering RNA, siRNA)转染、启动子荧光素酶报告基因活性测定、化学发光微粒子免疫法(Chemiluminescent Microparticle immunoassay, CMIA)以及染色质免疫共沉淀(chromatin immunoprecipitation, ChIP)等实验技术,联合类固醇激素及其受体拮抗剂、信号通路激动剂及其拮抗剂和转录因子抑制剂等工具药,研究了糖皮质激素对人类胎盘合体滋养层细胞芳香化酶的表达调控作用及其分子机制。研究结果1.人胎盘组织的免疫组织化学染色结果表明,芳香化酶主要在胎盘绒毛小叶的合体滋养层中大量表达,而绒毛小叶的细胞滋养层细胞、间质细胞和胎儿血管几乎无表达。此外,通过qRT-PCR和Western blotting的测定发现,芳香化酶的mRNA和蛋白水平随着细胞滋养层细胞向合体滋养层细胞的分化而不断增加。2. qRT-PCR和Western blotting测定结果表明,皮质醇(0.01-1μM,24h)能够剂量依赖性诱导人胎盘合体滋养层细胞芳香化酶mRNA和蛋白的表达。而且,EIA的测定结果表明,皮质醇(1μM,24h)也可以诱导胎盘合体滋养层细胞中DHEA向雌二醇的转化。3.免疫组织化学、普通PCR以及Western blotting的实验表明,人胎盘合体滋养层细胞表达大量的糖皮质激素受体(GR)；在此基础上,我们采用qRT-PCR、Western blotting以及GR siRNA转染细胞等实验方法研究发现,皮质醇(0.1μM,24h)对人胎盘合体滋养层细胞芳香化酶mRNA和蛋白表达的诱导作用能够被GR拮抗剂RU486 (1 μM)和GR siRNA(120 nM)阻断；此外,皮质醇对芳香化酶mRNA的诱导作用可以被蛋白质合成抑制剂放线菌酮CHX(10 μM)阻断,这些结果提示皮质醇对人胎盘合体滋养层细胞芳香化酶mRNA表达的诱导作用需要GR参与介导,但此介导作用可能是通过诱导其它蛋白合成间接实现的。4. qRT-PCR和Western blotting的检测结果表明,cAMP的类似物db-cAMP(100μM,24h)和腺苷酸环化酶的激动剂Forskolin (l00 μM,24h)都能够显著。促进胎盘合体滋养层细胞芳香化酶的表达；此外,我们利用EIA方法测定胞内cAMP水平发现,皮质醇(1μM,12h)可以显著增加胎盘合体滋养层细胞的胞内cAMP水平；进一步研究显示,PKA的抑制剂H89(10μM)以及Gαs的抑制剂NF449(20 μM)均可以阻断皮质醇(1μM,24h)对胎盘芳香化酶表达的诱导作用。这些结果提示,皮质醇对胎盘合体滋养层细胞中芳香化酶的表达调控是间接通过cAMP/PKA信号通路实现的。5.众所周知,糖皮质激素的经典作用需要通过细胞内的GR受体介导,于是我们推测在胎盘合体滋养层细胞中,糖皮质激素很可能通过GR诱导了与cAMP/PKA信号通路激活相关的激素的合成与分泌从而来促进胎盘芳香化酶的表达和雌激素的合成。已有报道,皮质醇可以通过GR诱导胎盘合体滋养层细胞CRH和hCG的表达与分泌,并且CRH和hCG的受体都能激活cAMP/PKA信号通路。因此,我们对CRH和hCG是否参与了皮质醇对人胎盘合体滋养层细胞芳香化酶的表达调控作用进行了研究。通过qRT-PCR测定发现,皮质醇(0.01-1μM,12h)可以剂量依赖性的诱导胎盘合体滋养层细胞CRH和hCG a与β亚基的表达,进一步用EIA和CMIA测定发现,皮质醇(1μM)处理胎盘合体滋养层细胞12小时后就可以显著增加胎盘合体滋养层细胞CRH和hCG的分泌,且该作用可以一直持续到24小时。而且,qRT-PCR、 Western blotting以及EIA测定结果表明,CRH受体的非选择性拮抗剂x-h-CRH (1 μM)和hCG抗体(1：100)单独都可以部分阻断皮质醇(1gM)对胞内cAMP水平和芳香化酶表达的诱导作用；当这两者联合用药时,则不仅可以降低胎盘合体滋养层细胞胞内cAMP的基础水平和芳香化酶的基础表达,还能进一步完全阻断皮质醇对cAMP水平和芳香化酶表达的诱导作用。这些结果提示CRH和hCG不仅通过cAMP/PKA信号通路共同维持着胎盘合体滋养层细胞芳香化酶的基础表达,而且还介导了皮质醇对胎盘合体滋养层细胞芳香化酶的诱导作用。6.由于胎盘芳香化酶基因可以利用不同的转录起始位点来进行转录,因此我们通过设计识别不同转录产物的特异引物进行PCR,研究发现胎盘合体滋养层细胞主要表达芳香化酶基因启动子PI.1的转录产物,并伴有少量启动子PI.2和P2a的表达产物,qRT-PCR检测结果还表明皮质醇(1μM,24h)对包含芳香化酶基因外显子I.1和包含外显子2a的转录产物具有诱导作用,而对包含芳香化酶基因外显子I.2的转录产物则无作用；此外,荧光素酶报告基因活性测定实验发现皮质醇(1μM,24h)和Forskolin (100 μM,24h)都可以显著诱导胎盘合体滋养层细胞芳香化酶基因-501bp启动子PI.1的活性,这说明皮质醇通过作用于芳香化酶基因启动子PI.1诱导了胎盘芳香化酶的表达。7.我们对芳香化酶基因启动子PI.1序列生物信息学分析发现,此序列上具有多个Sp1结合位点,因此进一步研究了Sp1在胎盘芳香化酶表达调控中的作用。用qRT-PCR和Western blotting的方法研究发现,皮质醇(1 μM)、 db-cAMP(100μM)和Forskolin (100 μM)处理细胞24h后都能够显著诱导胎盘合体滋养层细胞Sp1的表达,且皮质醇对Sp1的诱导作用也可以被H89以及NF449所阻断；此外,Sp1的拮抗剂光神霉素A (Mythramycin A, M,50μM)和Sp1的siRNA (60 nM)不仅能够抑制胎盘芳香化酶的基础表达,而且还能够抑制皮质醇和Forskolin对芳香化酶表达的促进作用。进一步用ChIP方法研究发现,皮质醇(1μM)以及db-cAMP(l00 μM)和Forskolin (100 μM)处理胎盘合体滋养层细胞24h都能促进Sp1与芳香化酶基因启动子PI.1的结合。同时,a-h-CRH (1 μM)以及hCG抗体(1：100)单独都可以部分阻断皮质醇对Sp1表达的诱导作用以及皮质醇对Sp1与芳香化酶基因启动子PI.1结合的促进作用；且当两者联合给药时,则不仅可以降低Sp1的基础表达以及Sp1与芳香化酶基因启动子PI.1基础结合,而且还可以进一步完全阻断皮质醇对Sp1的诱导作用以及皮质醇对Sp1与芳香化酶基因启动子PI.1结合的促进作用。这些结果说明,Sp1介导了皮质醇对胎盘合体滋养层细胞芳香化酶表达的诱导作用。皮质醇(1 μM Forskolin (100 μM)和db-cAMP (100 μM)除了可以增加Sp1与胎盘合体滋养层细胞芳香化酶基因启动子PI.1的结合外,还可以增加胎盘芳香化酶基因启动子PI.1上组蛋白3第九位赖氨酸的乙酰化(Acetyl H3K9)水平,并降低组蛋白3第九位赖氨酸的双甲基化(Dimethyl H3K9)水平,同时还能增加RNA聚合酶II(Pol Ⅱ)与芳香化酶基因启动子PI.1的结合,这说明胎盘芳香化酶启动子PI.1上组蛋白3的乙酰化和双甲基化也参与了皮质醇对胎盘芳香化酶基因的表达调控。结论我们的研究结果表明,人胎盘芳香化酶主要在胎盘合体滋养层细胞中表达,且随着胎盘滋养层细胞向合体滋养层细胞的融合而逐渐增加,此外,在合体滋养层细胞中,芳香化酶基因主要表达包含外显子I.1的转录产物。我们的研究结果还表明,在人类胎盘合体滋养层细胞中,皮质醇可以上调胎盘芳香化酶的表达,从而增加雌激素的合成。皮质醇通过结合其自身受体GR,进而诱导了胎盘合体滋养层细胞中CRH和hCG的合成与分泌,分泌到细胞外的CRH和hCG激活胎盘合体滋养层细胞胞内cAMP/PKA信号通路,该通路导致Sp1表达的增加以及Sp1与芳香化酶基因启动子PI.1的结合的增加,并改变芳香化酶基因启动子PI.1上组蛋白的表观遗传修饰,增强了芳香化酶基因启动子PI.1的转录活性,从而使得胎盘芳香化酶的表达上调。本研究提供了糖皮质激素上调人胎盘芳香化酶的表达从而促进雌激素合成的证据,为糖皮质激素参与人分娩启动提供了新观点,这些发现也可能为临床干预早产提供新的思路。
[Abstract]:According to the research background, according to data provided by the WHO, there are about 15 million premature infants worldwide each year, of which about 1 million infants died of premature birth complications and the number is still rising year by year. However, due to the lack of complete parturition mechanism in human childbirth, there has been a lack of effective means to prevent premature delivery. The analysis of the initiation mechanism of human childbirth has become one of the hot topics in the field of International Reproductive Biology in recent years. In most mammals including humans, the increasing estrogen in the end of pregnancy plays an important role in the process of childbirth. The estrogen induces the uterine myometrium to change from resting state to contraction. Delivery is prepared. Therefore, the study of the regulation of estrogen synthesis at the end of pregnancy is essential to elucidate the mechanism of birth start. Previous studies have shown that glucocorticoid is the key hormone in some animals such as sheep, which can promote the expression of placental cytochrome P450c17 hydroxylase, which catalyzes the pregnancy of placenta pregnancy. However, primates, including human placenta, lack P450c1 7 hydroxylase, and therefore can not make direct use of progestin to synthesize estrogen, instead, the human placenta can use the fetus and mother's kidney. Glandular dehydroepiandrosterone sulfate is a precursor, and a series of enzymatic reactions, especially aromatase, are used to produce estrogens. In human placenta, whether glucocorticoids can promote the synthesis of placental estrogens by affecting the expression of aromatase to participate in the process of human childbirth has not yet been reported. The human placental tissue and the placental trophoblast cells were used as the research model, using immunohistochemical staining, cultured in vitro placental trophoblast cells, real-time fluorescent quantitative PCR (quantitative real time PCR, qRT-PCR), Western blot hybridization (Western blotting), enzyme immunoassay (Enzyme immunoassay, EIA), small interference RNA. Ng RNA, siRNA) transfection, promoter luciferase reporter gene activity assay, chemiluminescent particle immunization (Chemiluminescent Microparticle immunoassay, CMIA) and chromatin immunoprecipitation (chromatin immunoprecipitation, ChIP), combined with steroid hormone and its receptor antagonist, signal pathway agonist and The effect of glucocorticoid on the expression of aromatase in human placental syncytiotrophoblast and its molecular mechanism. The results of immunohistochemical staining of 1. human placental tissues showed that aromatase was mainly in the syncytiotrophoblast of placental villi. In addition, the mRNA and protein levels of aromatase increased with the differentiation of cell trophoblast cells to syncytrotrophoblast cells and the results of.2. qRT-PCR and Western blotting measured by qRT-PCR and Western blotting. Cortisol (0.01-1 mu M, 24h) can induce the expression of aromatase mRNA and protein in human placental trophoblast cells in a dose-dependent manner. Moreover, the results of EIA assay showed that cortisol (1 u M, 24h) can also induce the conversion of DHEA to estradiol in placental chimeric cells by.3. immunohistochemistry, ordinary PCR and Western blotting. The results show that human placental syncytiotrophoblast cells express a large number of glucocorticoid receptor (GR). On this basis, we use qRT-PCR, Western blotting and GR siRNA transfected cells to study the results of the results that the induction of cortisol (0.1 mu M, 24h) on the expression of the aromatase mRNA and protein expression of human placental syncytiotrophoblast cells can be G. R antagonists, RU486 (1 u M) and GR siRNA (120 nM), are blocked. In addition, the induction of cortisol to aromatase mRNA can be blocked by the protein synthesis inhibitor actinomycete CHX (10 u M). These results suggest that the inducement of cortisol to the expression of aromatase mRNA for human placental syncytiotrophoblastic cells requires GR involvement, but this mediating action The results of.4. qRT-PCR and Western blotting, which may be indirectly achieved by inducing other protein synthesis, showed that cAMP analogues db-cAMP (100 mu M, 24h) and adenylate cyclase, Forskolin (l00 mu M, 24h), were all significant. The intracellular cAMP level showed that cortisol (1 mu M, 12h) could significantly increase the intracellular cAMP level of the placental syncytiotrophoblast cells. Further studies showed that PKA inhibitor H89 (10 mu M) and G alpha s inhibitor NF449 (20 mu M) could block the induction of cortisol (1 mu M) on the expression of placental aromatase. These results suggest that the skin of the placental aromatase is induced. The regulation of aromatase in placental syncytiotrophoblast cells is known as.5., which is known indirectly through the cAMP/PKA signaling pathway. The classic role of glucocorticoids is mediated by intracellular GR receptors. Therefore, we speculate that glucocorticoids are likely to be induced by GR in placental syncytiotrophoblast cells by GR It has been reported that cortisol can induce the expression and secretion of CRH and hCG in the placental syncytiotrophoblast cells by GR, and that both CRH and hCG receptors can activate cAMP/PKA signaling pathways. Therefore, we are on CRH and hCG. The effect of cortisol on the regulation of the expression of aromatase in human placental syncytiotrophoblast was studied. The qRT-PCR assay showed that cortisol (0.01-1 M, 12h) could induce the expression of CRH and hCG A and beta subunits in placental chimeric trophoblast cells in a dose-dependent manner, and were detected by EIA and CMIA, and the treatment of cortisol (1 mu M). The placental syncytrotrophoblast cells can significantly increase the secretion of CRH and hCG in the placental trophoblast cells for 12 hours, and the effect can last for 24 hours. Moreover, qRT-PCR, Western blotting and EIA results show that the non selective antagonist of CRH receptor x-h-CRH (1 u M) and hCG antibody (1:100) can be partially isolated. The inhibitory effect of cortisol (1gM) on the intracellular cAMP level and aromatase expression, when the combination of these two drugs, can not only reduce the basal level of intracellular cAMP and the basic expression of aromatase in the placental syncytiotrophoblast cells, but also further completely block the induction of cortisol on the level of cAMP and aromatase expression. These results suggest that CRH and hCG not only maintain the basic expression of aromatase in the placental syncytiotrophoblastic cells through the cAMP/PKA signaling pathway, but also mediate the effect of cortisol on the aromatase of the placental syncytiotrophoblast cells,.6., as the placental aromatase gene can be transcribed with different transcriptional starting sites. So by designing specific primers that identify different transcripts for PCR, we found that placental syncytiotrophoblast cells mainly express the transcriptional products of aromatase gene promoter PI.1, accompanied by a small number of promoter PI.2 and P2a expression products. The qRT-PCR detection results also indicate that cortisol (1 mu M, 24h) is outside the inclusion of aromatase genes. The transcriptional products of the exon I.1 and the exon 2A were induced, while the transcriptional products containing the aromatase exon I.2 had no effect. In addition, the luciferase reporter gene activity assay found that cortisol (1 mu M, 24h) and Forskolin (100 mu M, 24h) could significantly induce the aromatase base of the placental syncytiotrophoblast cells. The activity of the -501bp promoter PI.1 suggests that cortisol induced the expression of placental aromatase by the role of the aromatase gene promoter PI.1.7.. We found that there are multiple Sp1 binding sites on the PI.1 sequence of the aromatase gene promoter, and thus further studies the Sp1 aromatase in the placenta. The expression of qRT-PCR and Western blotting showed that cortisol (1 mu M), db-cAMP (100 u M) and Forskolin (100 mu M) can significantly induce the expression of Sp1 of the placental syncytiotrophoblast cells, and the induction of cortisol to Sp1 can also be blocked by H89 and the antagonists. A (Mythramycin A, M, 50 mu M) and siRNA (60 nM) of Sp1 not only inhibit the basic expression of placental aromatase, but also inhibit the promotion of cortisol and Forskolin on the expression of aromatase. Further ChIP methods have been used to study the treatment of Corticosterol (1 mu M) and db-cAMP (100 mu). The placental syncytiotrophoblast cell 24h can promote the binding of Sp1 with the aromatase gene promoter PI.1. At the same time, a-h-CRH (1 mu M) and hCG antibody (1:100) alone can partially block the induction of cortisol to Sp1 expression and the promotion of cortisol to the combination of Sp1 and aromatase promoter PI.1. The drug can not only reduce the basic expression of Sp1 and the combination of Sp1 with the aromatase gene promoter PI.1, but also further completely block the induction of cortisol to Sp1 and the promotion of cortisol on the binding of Sp1 to the aromatase gene promoter PI.1. These results suggest that Sp1 mediates the combination of cortisol to placenta. The induction of aromatase expression in the trophoblast cells. Cortisol (1 M Forskolin (100 u M) and db-cAMP (100 mu M) can increase the binding of Sp1 to the PI.1 of the aromatase gene of the placental syncytiotrophoblast cell, and increase the acetylation of the histone 3 and ninth lysine (A) on the placental aromatase gene of the promoter PI.1 (A). Cetyl H3K9) level and decrease the level of dimethylation (Dimethyl H3K9) of the histone 3 ninth bits lysine and increase the binding of RNA polymerase II (Pol II) with the aromatase gene promoter PI.1, which indicates that the acetylation and dimethylation of histone 3 on the placental aromatase promoter are also involved in cortisol to placental aromatase. Conclusion our results show that the human placental aromatase is mainly expressed in the placental syncytial trophoblast cells and gradually increases with the fusion of placental trophoblast cells to the syncytrotrophoblast cells. In addition, in the syncytial cells, the aromatase gene mainly expresses the transcriptional production of exon I.1. Our results also show that in human placental syncytiotrophoblast cells, cortisol can increase the expression of placental aromatase and increase the synthesis of estrogen. Cortisol, by combining its own receptor GR, induces the synthesis and secretion of CRH and hCG in the placental syncytiotrophoblast cells, and secretes CRH and hCG out of cells. Activating the intracellular cAMP/PKA signaling pathway of the placental syncytial cells leads to an increase in the expression of Sp1 and the increase of the binding of Sp1 to the aromatase gene promoter PI.1, and changes the epigenetic modification of the histone on the aromatase gene promoter PI.1, and enhances the transcriptional activity of the aromatase gene promoter PI.1, thus making it possible to increase the transcriptional activity of the aromatase gene promoter PI.1. This study provides evidence that glucocorticoids up the expression of human placental aromatase to promote estrogen synthesis, providing new ideas for the involvement of glucocorticoids in human birth, and these findings may also provide new ideas for clinical intervention in preterm labor.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.21

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