【作者】 李苑；

【导师】 古航； 倪鑫；

【作者基本信息】 第二军医大学 ， 妇产科学， 2012， 硕士

【摘要】 研究证实，前列腺素（prostaglandin，PG）作为诱发子宫收缩的主要内源性因子，通过促进宫颈成熟、诱发破膜、启动和维持子宫肌收缩等作用，参与了人类的分娩过程。前列腺素合成关键酶为前列腺素H合成酶（prostaglandin H synthetase，PGHS），而其代谢主要通过NAD依赖的15－羟基前列腺素脱氢酶（15-hydroxyprosglandindehydrogenase,PGDH）转化为无活性的15－酮基代谢产物。妊娠期间前列腺素主要来源于宫内组织，如羊膜、绒毛膜和胎盘等。研究证明羊膜高表达PGHS，可以合成高浓度PG，而绒毛膜不仅表达PGHS，还高表达PGDH，因此在妊娠期间绒毛膜作为一个代谢屏障调控羊膜及其自身产生的前列腺素向子宫肌的转运。因此，妊娠期间羊膜和绒毛膜产生的前列腺素主要被绒毛膜的PGDH所降解，到达子宫的量很少。但临近分娩时，一方面，羊膜和绒毛膜PGHS表达增加，另一方面，绒毛膜PGDH表达减少，导致胎膜来源的前列腺素到达子宫的量显著增加。研究证实早产患者绒毛膜PGDH显著地低于同孕龄正常孕妇绒毛膜，说明绒毛膜PGDH表达减少可能是子宫发动收缩的重要原因。虽然目前关于绒毛膜PGDH的调节机制尚未完全阐明，但近年来的研究提示绒毛膜PGDH的表达主要受到宫内产生的生物活性物质的调节，如糖皮质激素和孕激素，孕激素促进绒毛膜PGDH的表达，而糖皮质激素的作用则与之相反——抑制PGDH的表达，即两者在调节前列腺素代谢方面呈拮抗作用，而其作用机制尚不明确。研究发现，孕激素均可以和GR、PR结合，而生理浓度GC仅和GR结合发挥调节作用，且绒毛膜滋养层细胞既可表达GR，也可表达两型PR：PRA和PRB。那么上述GC和孕激素对绒毛膜PGDH调节的拮抗作用究竟是通过哪型受体实现的呢？即GR、PRA和PRB等各型受体在孕激素、GC调节绒毛膜上PGDH过程中发挥何种调节作用，目前并不清楚。因此课题的第一部分利用原代培养的绒毛膜滋养细胞，首先通过构建siRNA和受体表达载体等方法分别使使细胞GR、PR的表达敲低或过度表达，明确GC和孕激素调控绒毛膜PGDH表达的受体机制；进而在此基础上，构建含不同长度的PGDH启动子序列的报告基因转染细胞，推测GC和孕激素调控PGDH表达的关键序列。过氧化物酶体增殖体激活受体（peroxisome proliferation-activated receptors，PPARs）属于配体激活性核激素受体超家族，存在三个亚型：PPARα、PPARβ和PPARγ，PPARs各亚型在人绒毛膜上均有表达。研究证实PPARs不仅参与滋养层细胞分化、胚胎植入、脂质和葡萄糖代谢及血管发生等过程，还与分娩过程密切相关。不同子宫收缩状态或分娩进展时，PPARs各亚型的表达和DNA结合活性有差异。PPARs还可以通过下调胎膜COX-2和sPLA2的表达来降低前列腺素的浓度，从而维持妊娠期间子宫的静息状态，说明PPARs参与前列腺素合成的调节，那么PPARs是否影响PG的代谢即是否调节绒毛膜PGDH的表达和活性？本课题第二部分利用原代培养的人绒毛膜滋养层细胞，研究不同PPARs对绒毛膜PGDH表达的调节作用及其分子机制。硫化氢（hydrogen sulphide，H2S）是人类发现的第三种气体信号分子，具有舒张血管平滑肌、缓解肺动脉高压、抗心肌缺血、参与炎症反应等多种功能。现在对于H2S的研究还主要集中在心血管系统、神经系统和炎症反应等方面，而在生殖系统中关于H2S研究才刚刚起步。Patel等人在2009年首次发现人类宫内组织如子宫肌、绒毛膜及胎盘中均有CBS和CSE的表达，并可以产生内源性H2S，低氧时人胎盘H2S生成增加，从而推测H2S可能参与子痫前期的发病过程，但并未进一步研究。因此本课题第二部分，我们收集正常足月临产、正常足月未临产及妊娠期高血压疾病（未临产）的绒毛膜及羊膜组织，检测其中的H2S生成率及H2S合成酶CSE、CBS在不同妊娠状态胎膜（包括绒毛膜和羊膜组织）上的表达。主要实验结果如下：第一部分、糖皮质激素和孕激素调节绒毛膜前列腺素代谢酶PGDH的机制研究1.免疫组织荧光技术证实绒毛膜滋养细胞上有PR的表达。临产分娩所得绒毛膜组织PGDH和PRB mRNA和蛋白表达量显著低于未临产分娩所得绒毛膜组织表达量；而临产分娩所得绒毛膜组织PRA和GR mRNA和蛋白表达量显著高于未临产分娩所得绒毛膜组织表达量，提示PR和GR表达的改变可能参与了分娩启动过程。统计软件分析表明，临产和未临产绒毛膜组织GR、PGDH蛋白的表达呈负相关，提示糖皮质激素可能通过GR抑制了绒毛膜上PGDH的表达。2.用人工合成的糖皮质激素Dexamethasone（Dex）、孕激素P4及孕激素合成酶3β-HSD抑制剂Trilostane（Tril）处理细胞，发现Dex和Tril呈剂量依赖性方式下调绒毛膜滋养PGDH mRNA和蛋白表达及其酶活性；而P4则剂量依赖性上调PGDHmRNA和蛋白表达及其活性。提示内源性孕激素对绒毛膜PGDH的表达可能起维持作用，外源性孕激素可以促进其表达和活性，糖皮质激素作用与之相反。3. GR干扰或PRB过表达后，PGDH的蛋白表达及其酶活性均上调，而PRA过表达后PGDH的蛋白表达及其酶活性下调，提示A、B两型PR受体对PGDH的蛋白表达作用相反。GR干扰和PRB过表达后，P4处理组PGDH蛋白表达及其酶活性进一步上调，提示孕激素通过与PRB结合上调PGDH的蛋白表达及其酶活性。而在PRA过表达后，P4表现出对PGDH的抑制效应，提示P4与PRA结合抑制了PGDH的表达。当过表达PRA或PRB后，Dex对PGDH蛋白表达和酶活性的抑制效应均存在，而GR干扰后，Dex对PGDH蛋白表达和酶活性的抑制效应消失，提示Dex主要通过与GR结合发挥对PGDH的抑制作用，而可能并不通过与PR结合发挥效应。PR干扰或GR过度表达后，PGDH蛋白的基础表达及活性均下调，在此基础上，分别用Dex和P4处理细胞， PGDH蛋白表达和活性进一步下调，提示Dex和P4均能通过与GR结合抑制PGDH蛋白表达和活性；而用Tril处理细胞，PGDH蛋白表达和活性与转染空载体并用Tril处理细胞相比，并无明显差异。提示内源性孕激素主要维持PGDH的蛋白表达和酶活性。总结：P4与PRB结合促进绒毛膜上PGDH的表达，与PRA、GR结合抑制其表达，Dex对绒毛膜PGDH的抑制作用主要通过GR实现，可能并不与PR结合。4.将含有不同长度PGDH基因的荧光素酶报道基因表达载体转染原代绒毛膜滋养层细胞，结果发现，Dex和Tril对含有2368bp和2038bp PGDH启动子的报道基因转录活性有明显地抑制作用，而P4则呈促进作用；Dex、 P4和Tril对含有1024bp、388bp和233bpPGDH启动子的报道基因转录活性三者都没有调节作用。提示2368bp至1024bp之间含有Dex、P4与Tril调节PGDH基因表达的关键序列。第二部分、不同亚型过氧化酶体增殖物激活受体对绒毛膜PGDH表达的调节作用及其机制1．用不同浓度的PPARα、PPARβ、PPARγ各特异性激动剂GW7467、GW0742、Rosiglitazone（10-9～10-6M）及其各特异性拮抗剂GW6471、GSK0661、GW9662（10-9～10-6M）单独处理细胞，结果表明不同亚型PPARs均能促进绒毛膜PGDHmRNA和蛋白的表达，且以PPRγ的促进作用最强；各亚型的特异性拮抗剂均能剂量依耐性抑制PGDH mRNA和蛋白的表达，且特异性拮抗剂均能逆转相对应的激动剂对PGDH mRNA和蛋白表达的促进作用。提示PPARs激动剂可以促进绒毛膜上PGDH的mRNA和蛋白表达2.分别干扰细胞PPARα、PPARβ、PPARγ的表达后，PGDH蛋白表达均下降，再给予相对应的特异性激动剂GW7467、GW0742、Rosiglitazone（10-7M）处理细胞，GW7467、GW0742、Rosiglitazon不能继续上调绒毛膜PGDH的蛋白表达。说明上述药物上调绒毛膜PGDH表达的作用是分别通过PPARα、β、γ实现的。提示PPARs对PGDH表达的上调作用并非激动剂的药物作用，可能是PPARs本身的效应。3.用RXR激动剂9-cis-RA处理细胞，结果发现9-cis-RA呈剂量依耐性促进PGDH mRNA和蛋白表达，且对PPARs上调PGDH mRNA表达的作用具有增强的趋势，提示PPARs对PGDH表达的调节作用可能是通过与RXR形成异源二聚体而实现。4.给予蛋白合成抑制剂CHX（10-5M）预处理细胞1小时，再给予GW7467、GW0742、Rosiglitazone处理，结果PPARs激动剂仍能上调PGDHmRNA的表达。提示PPARα、β、γ对绒毛膜PGDH mRNA表达的调节作用不需要合成新蛋白，可能是对PGDH的直接作用。5. PPARs激动剂和mRNA合成抑制剂DRB先后处理细胞不同时间，结果显示PPARs激动剂绒毛膜细胞PGDHmRNA半衰期，说明不同亚型PPARs对PGDH mRNA表达的上调作用，不是通过影响PGDH mRNA的稳定性实现的。6.收集正常足月临产、未临产绒毛膜组织，检测PPARs三种亚型各自的mRNA和蛋白的表达。结果显示未临产分娩所得绒毛膜组织PPARγ mRNA和蛋白表达量显著低于临产分娩所得绒毛膜组织，而PPARα、β mRNA和蛋白表达则无明显差异。临产后PGDHmRNA和蛋白表达是显著下降的，提示在体情况下，妊娠晚期PPARs中可能由于γ亚型的表达下降引起绒毛膜PGDH表达的减少，宫内组织局部前列腺素浓度升高，这可能是PPARγ参与分娩启动的机制之一。第三部分、硫化氢及其合成酶在不同妊娠状态胎膜的表达1．免疫组织化学染色显示人绒毛膜组织及羊膜组织均表达H2S合成酶--CBS和CSE，且绒毛膜上主要表达于合体滋养层细胞，而羊膜主要表达于上皮细胞。2．收集正常足月临产、正常足月未临产及未临产子痫前期（preeclampsia，PE）的绒毛膜及羊膜组织，检测其H2S生成率及其合成酶CSE、CBS在胎膜（包括绒毛膜和羊膜组织）上蛋白和mRNA的表达。结果显示，正常足月临产组胎膜硫化氢生成率及CSE、CBS蛋白和mRNA的表达较正常未临产组明显下降，PE患者胎膜CBS和CSE mRNA及蛋白表达、H2S的生成速率较正常者也显著减少。提示在人胎膜H2S可能参与分娩启动和子痫前期的病理过程。结论：1.孕激素和糖皮质激素可以通过与不同受体结合，发挥对PGDH的不同调节作用，二者共同调节绒毛膜上PGDH的表达，从而影响绒毛膜前列腺素的浓度，这可能是孕激素和糖皮质激素参与妊娠维持和分娩启动的机制之一。糖皮质激素和孕激素对PDGH的这些调节作用可以通过影响PGDH的启动子来实现，启动子-2368至-1024之间这段碱基序列含有PGDH基因表达的关键序列。2. PPARs三种亚型均可促进绒毛膜滋养细胞上PGDH的表达，这种调节作用无需新蛋白参与，也不影响PGDH mRNA的稳定性，而是通过直接调节PGDH的基因转录来实现的。在临产的绒毛膜组织中PPARγ的mRNA及蛋白表达明显下降，而PPARα、PPARβ的表达无明显差异，提示绒毛膜上PPARγ亚型临产前后的变化可能与分娩启动过程相关。3． H2S及其合成相关酶CBS、CBS在不同妊娠状态的人胎膜组织中表达不同，人胎膜H2S可能参与分娩启动和子痫前期的发病。更多还原

【Abstract】 Prostaglandin(PG) produced within the intrauterine tissues play a central role in theinitiation and progression of labor in human. It has been shown that PG maintain uterineand placental blood flow, regulate the cervical ripening, membrane rupture and induceuterine contractility. PG is synthetized by Prostaglandin-H-synthase (PGHS), whilemetabolised by15-hydroxyprostaglandin dehydrogenase (PGDH). Not only PGHS but alsoPGDH is expressed in chorion during pregnancy. However a very high concentration ofPGDH has been localized in chorion.So high level PG is synthetized in amnion,metabolised in chorion,and after acrossing full thickness membranes becomes lower inplacenta and myometrium. Several reports have indicated that chorion has been describedas a protective barrier to prevent the passage of primary PG synthesized within the amnionor chorion from reaching the myometrium and stimulating the onset of preterm or termdelivery. But the regulation of chorion PGDH has not yet been elucidated.Although glucocorticoids and progesterone were demonstrated that could regulate theexpression and activity of PGDH in chorion via bingding glucocorticoid receptor(GR) orprogesterone receptor(PR),the mechanism of their effect is unclear. Chorion expresses bothGR, PRA and PRB—two types of PR.We conducted expreriments to determine thereceptors mediating the effect of GC and P4on PGDH in chorion.We treated choriontrophoblast cells with dexamethasone, progesterone and trilostane (an inhibitor of3β-HSD), to determine PGDH activity and expression. To examine the role of GR and PR onPGDH, we constructed GR siRNA, PR siRNA and their overexpression plasmid.Peroxisome proliferator-activated receptors (PPARs) are transcription factorsbelonging to the ligand-activated nuclear hormone receptor superfamily,and present threeisotypes:α,β andγ. High expression of PPARs has been localized in human intrauterinetissues,such as amnion,chorion,placenta.PPARs are known not only to be involved in thephysiological and pathological events occurring during the placentation, but also contributeto the control of parturition in humans. term human labour is associated with changes inexpression and activity of PPAR isoforms. The production of prostaglandins by the fetalmembranes induces the contraction of the myometrium during labor. This generation ofuterotonic prostaglandins correlates with the increased COX-2activity and the increasedsecretory sPLA2mRNA, proteins and activities. By inhibiting the production of theCOX-2and sPLA2in fetal membranes, PPARs promotes the quiescence of the uterusduring gestation.In the second part of this subject, We culture chorion trophoblast cells to examine the regulation and its mechanism of PPARs on PGDH.The gas hydrogen sulfide (H2S) is emerging as a novel regulator of importantphysiologic functions such as arterial diameter, blood flow and leukocyte adhesion. Inaddition, it may have anti-inflammatory and anti-apoptotic effects. H2S has recentlyattracted much interest as a new gaseous signal molecule.However there is few reportsabout physiological and pathological events caused by H2S in the reproductive system.In2009,Patel et al published the first study to report the detection of both CBS and CSE inhuman intrauterine tissues and the production of H2S by these tissues.They considered thatendogenously produced H2S could possibly have a role in the pathology of pre-eclampsia.However further investigation of the role of H2S in the reproductive system is required.Inthis part,we examined the expression of H2S synthesis enzyme CSE,CBS in fetalmembranes (chorion and amnion).The main results:1.Regulation of PGDH by Glucocorticoid and Progesterone in human Chorion1). In nonlabor chorion tissue, PGDH/PRB mRNA and protein expression issignificantly higher than those in labor tissue,however PRA/GR mRNA and proteinexpression in nonlabor chorion tissue is much lower than in labor. Moreover, Statisticalanalyses showed that there is a negative correlation between the expression of PGDH andGR.2). PGDH protein and mRNA expression levels were detected by Western blot andreal-time quantitative PCR assay respective. DEX and Trilostane dose dependentlydown-regulated the mRNA and protein expression of PGDH. Treatment of chorion cellswith P4caused a dose-dependently increase in the mRNA and protein expression ofPGDH.3) Using RNA interference and over-expression technology to prevent and promoteGR and PR expression respectively,then treated cells with Dex,Tril and P4, PGDH proteinlevels were detected by Western blot.PGDH activity and protein expression is decreased byPR interference and GR/PRA over-expression,while is increased by GR interference andPRB over-expression.Dex decrease effect is also exist moderately with GR interferenceand is doubled with GR over-expression.PR interference and PRa/PRb over-expressioncannot change the effect strength of Dex on PGDH. P4promoted the PGDH activity andprotein expression further with GR interference and PRB over-expression,while inhibitedwith PR interference and GR/PRA over-expression.Trilostane down-regulated the protein level and activity of PGDH.4) With the regulation of steroids on chorion PGDH gene transcription activity ofrelated D NA sequences identified by5’ terminal deletion methods obtained with differentlength of PGDH gene luciferase reporter gene expression vector. Dex, P4and Tril activityhas obvious inhibition or activation effect to PGDH promoter transcription of a reportergene containing-2368bp and-2038bp, and has no moderating effect activity to the onecontaining-1024bp,-388bp and-233bp.2.Regulation and mechanism of PGDH by Peroxisome proliferator-activatedreceptors (PPARs) in human Chorion1) A increase in the mRNA and protein expression of PGDH is caused by thetreatment of chorion cells with PPARα、PPARβ、PPARγ agonist GW7467、GW0742、Rosiglitazone（10-9～10-6M）and a decrease is caused by the antagonist GW6471、GSK0661、GW9662（10-9～10-6M）.These increase effects could be blocked by theantagonists.2) Using RNA interference technology to prevent PPARα、PPARβ、PPARγ expressionrespectively,then treated cells with GW7467、GW0742、Rosiglitazone, PGDH proteinlevels were detected by Western blot. PGDH protein expression is decreased by PPARsinterference.However GW7467, GW0742and Rosiglitazon can not continue to increasethe PGDH protein expression.The results showed that the upregulation of PPARs onPGDH expression is not an drug effects of agonists,but is the effects of PPARs own.3)RXR ligand9-cis-RA alone could increase the expression of PGDH in the chorioncells. And9-cis-RA potentiated the effect induced by PPARs agonists.4) Cells were treated with PPARs agonists in the absence and presence of CHX, aprotein synthesis inhibitor. It was found that GW7467,GW0742and Rosiglitazone wereequally effective in modulating PGDH mRNA in the absence and presence of CHX,indicating that denovo protein synthesis was not required.4) To elucidate the molecular mechanisms by which PPARs regulated PGDH mRNA,cells were treated with DRB, an inhibitor of mRNA synthesis. The results showed thatGW7467,GW0742and Rosiglitazone did not alter the half-life of PGDH mRNA.6) Western blot hybridization and real-time PCR showed that the expression ofPPARγ were significantly lower in labor chorion tissue compared with those in nonlabor,while the expression of PPARα and PPARβ had no change. Results suggested that PPARγinvolve in maintain of PGDH expression in chorion. 3. Expression of synthetases responsible for H2S production in huamanlabor,nonlabor and preeclampsia fetal membranes1). Both H2S synthetases,CBS and CSE，express in human amnion epithelial cells andchorion trophoblast cells showed by immunochemistry．2). RT-PCR and Western-blot were used to define the mRNA and protein expressionof CSE and CBS.To define the real-time kinetics of H2S production by fetal membranes，we used miniaturized H2S micro-respiration sensor to measure its production.Resultsshowed that lower level of H2S production,mRNA and protein expression of CSE and CBSin both labor chorion and amnion compared with nonlabor tissue,and higher level innormal chorion and amnion compared with preeclampsia tissue. It suggests that H2S mayinvolves in pregnancy progress and pregnant blood pressure control.Conclusion:1.In vitro human chorion trophoblast cells, the endogenic Progesterone maintain theexpression of PGDH. Howeve the regulation of exogenetic Progesterone on PGDH isdifferent via PR or GR. Exogenetic Progesterone promotes PGDH via PRb mainly, whileinhibit PGDH via GR and PRa. Glucocorticoid inhibit PGDH via GR. They worktogether to contribute to the normal concentration of PGDH in chorion.And there is areporter gene tips-2038to-1024between the base sequence containing the key sequenceof Dex, P4and Tril regulation of PGDH gene expression.2. PPARs modulate the PGDH expression in human chorion trophoblast cells, theseeffects are mediated primarily at transcriptional level. PPARγ involve in maintain ofPGDH expression in chorion tissue.3. H2S and its synthetic enzymes CBS, CBS expressed significantly different in humanfetal membranes of different pregnancy status.H2S may be involved in the parturition andthe of pathological process of preeclampsia.更多还原