【作者】 何平；

【导师】 倪鑫；

【作者基本信息】 第二军医大学 ， 生理学， 2011， 博士

【摘要】 糖皮质激素（glucocorticoid, GC）在妊娠期间起着极其重要的作用,不仅调节各种胎盘激素的合成和释放,还促进胎儿器官特别是肺、肠道和脑等的成熟[1-3]。但是胎儿过早、过多的接触GC可导致胎儿宫内发育迟缓（intrauterine growth restriction, IUGR）,与妊娠期高血压（pregnancy-induced hypertension, PIH）的发生发展也密切相关[4,5]。妊娠期间母体血液中GC浓度随孕期不断升高,是胎儿循环的5到十倍,且GC是甾体激素,可自由通过细胞膜,那么是如何保证在妊娠期间胎儿发育所需的一个适宜的GC环境呢?大量研究证实妊娠期间胎盘高表达的GC代谢酶——11β-羟基类固醇脱氢酶2（11β-hydroxysteroid dehydrogenase type 2,11β-HSD2）在其中发挥了关键作用,通过将有活性的GC代谢为无活性的代谢产物,以保证胎儿发育所需的适宜的GC环境,由此在胎盘局部起到功能性屏障作用^[6,7]。若胎盘11β-HSD2表达或功能异常,均可导致母体循环中高浓度GC通过胎盘进入胎儿循环,使胎儿过早、过多地接触GC,从而影响胎儿发育[8]。目前研究证实孕激素、NO、缺氧等可影响胎盘11β-HSD2的表达和活性[9],但关于胎盘11β-HSD2表达和活性的调控机制尚未完全阐明。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（或PPARδ）和PPARγ1-3]。研究发现人胎盘PPARs三个亚型均有表达,并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,与胎盘胎儿的发育密切相关^[11]。但关于各种配体激活PPARs后,通过调控哪些因素而参与胎盘胎儿发育过程并不清楚。PPARs的天然配体主要来源于饮食和机体的代谢产物^[12],研究表明限制动物蛋白摄入量或孕期营养不良可使胎盘11β-HSD2表达减少^[13]或活性降低^[14],但具体机制尚不清楚。因此课题的第一部分利用原代培养的人胎盘滋养层细胞,研究不同PPARs对11β-HSD2表达和活性是否有调节作用及其分子机制;收集正常和PIH孕妇所分娩的胎盘组织,检测PPARs三个亚型和11β-HSD2 mRNA及蛋白的表达。硫化氢（hydrogen sulphide,H₂S）是人类发现的第三种气体信号分子,在机体心血管系统、神经系统生理活动及病理改变中发挥重要作用^[15]。研究表明人类胎盘组织表达H₂S合成酶——CBS和CSE,并可生成H₂S^[16],但关于它们的具有功能及其调节并不清楚。胎盘作为妊娠期所特有的器官,也是体内最大的内分泌器官之一,可合成、分泌多种激素及其代谢酶、细胞因子等。其中胎盘合成和分泌的促肾上腺皮质激素释放激素（corticotropin-releasing hormone, CRH）不仅影响胎儿的发育与成熟、参与分娩启动,且与早产、IUGR、PIH等疾病有关^[17-19]。研究证实气体信号分子在CRH表达的调节中起着重要作用,如NO、CO、H₂S均可抑制下丘脑释放CRH,NO抑制胎盘滋养层细胞CRH的释放^[20,21]。因此本课题第二部分,利用免疫组化方法观察H₂S合成酶在胎盘组织中的表达定位;利用原代培养的人胎盘滋养层细胞,检测H₂S是否影响CRH mRNA、多肽的合成及其分泌;研究PPARs是否影响H₂S合成酶的表达和CRH mRNA、多肽的合成及其分泌;收集正常和PIH孕妇所分娩的胎盘组织,检测H₂S合成酶的表达和H₂S的生成。实验结果如下:第一部分不同亚型PPARs对胎盘11β-HSD2表达的调节作用及其机制1、定量PCR、western印迹杂交和放射酶活性测定结果显示PPARα特异性激动剂GW7647呈剂量依赖性方式抑制11β-HSD2 mRNA和蛋白表达及其氧化酶活性,PPARγ特异性激动剂罗格列酮则作用相反——呈促进效应,二者均在10-6M达到最大作用。上述作用可分别被PPARα特异性拮抗剂GW6471和PPARγ特异性拮抗剂GW9662逆转,说明PPARs不同亚型对胎盘11β-HSD2表达和活性具有不同的调节作用;2、蛋白合成抑制剂CHX不影响GW7647和罗格列酮对11β-HSD2的调节作用,提示上述PPARs对11β-HSD2 mRNA表达的调节作用不是通过影响其他蛋白的表达的间接作用,而是直接作用于11β-HSD2;3、RXRs配体9-顺-维甲酸单独作用可上调胎盘11β-HSD2 mRNA和蛋白表达,且对罗格列酮上调胎盘11β-HSD2表达的作用有增强的趋势;利用RXRasiRNA干扰胎盘RXRα表达后,可逆转罗格列酮调节胎盘11β-HSD2表达的效应。以上说明PPARγ对胎盘11β-HSD2的调节作用可能是通过和RXRα形成异源二聚体这个经典途径实现的。9-顺-维甲酸对GW7647下调11β-HSD2表达的作用没有影响,且干扰胎盘RXRα表达后,PPARα的调节效应依然存在,提示RXRα不参与PPARα对胎盘11β-HSD2的调节过程;4、mRNA合成抑制剂5,6-二氯-1-β-D呋喃核糖苯并咪唑（5,6-dichlorobenzimidazole 1-β-D-ribfuranoside, DRB）不影响GW7647和罗格列酮对11β-HSD2的调节作用,说明上述PPARs对11β-HSD2 mRNA表达的调节作用,不是通过改变11β-HSD2 mRNA的稳定性实现的;5、将含11β-HSD2基因启动子的荧光素酶报道基因瞬时转染原代培养的人胎盘滋养层细胞,结果显示GW7647抑制11β-HSD2的基因转录,其作用可被GW6471所逆转;罗格列酮则促进11β-HSD2的基因转录,GW9662可逆转此上调作用。以上结果说明PPARα、PPARγ分别抑制和促进胎盘11β-HSD2的基因转录;6、收集正常妊娠和PIH患者分娩的胎盘组织,结果显示PIH患者胎盘11β-HSD2、PPARγmRNA及蛋白表达较正常者显著减少,而PPARα和PPARβmRNA和蛋白表达较正常者显著增加。统计学分析结果显示11β-HSD2与PPARβ之间呈负相关、11β-HSD2与PPARγ之间具有正相关,提示胎盘不同亚型PPARs参与胎盘11β-HSD2表达的精细调控过程。第二部分不同亚型PPARs对胎盘H₂S合成酶表达的调节作用及其意义1、免疫组织化学染色显示人胎盘组织表达H₂S合成酶——CBS和CSE,且主要表达于合体滋养层细胞;2、利用原代人胎盘滋养细胞,用不同浓度的L-半胱氨酸或/和CSE抑制剂PAG/CBS抑制剂AOAA、D-半胱氨酸及NaHS处理细胞。结果显示L-半胱氨酸及NaHS抑制胎盘细胞CRH mRNA表达和多肽水平,PAG或AOAA均可逆转L-半胱氨酸的抑制作用,而D-半胱氨酸不影响胎盘细胞CRH mRNA表达和多肽含量。以上结果说明L-半胱氨酸在胎盘H₂S合成酶作用下生成H₂S,进而抑制胎盘细胞CRH的合成和分泌。3、利用原代人胎盘滋养细胞,给予PPARs各亚型特异性激动剂或/和拮抗剂处理。结果显示PPARγ特异性激动剂GW7647和PPARβ特异性激动剂GW0742抑制CSE、CBS mRNA和蛋白表达,作用可分别被各自特异性拮抗剂GW6471、GSK0660所逆转;PPARγ特异性激动剂罗格列酮促进CSE、CBS的表达,此作用也可为其阻断剂GW9662所逆转,说明PPARs不同亚型对胎盘H₂S合成酶的表达具有不同的调节作用;4、利用原代人胎盘滋养细胞,用PPARs各亚型特异性激动剂或/和拮抗剂处理细胞。结果显示GW7647和GW0742剂量依赖性地促进胎盘细胞CRH mRNA表达、CRH多肽的合成,而不影响CRH多肽的分泌量,GW6471、GSK0660可分别逆转上述作用;罗格列酮则剂量依赖性方式抑制胎盘细胞CRH mRNA表达、CRH多肽的合成和分泌量,GW9662可逆转此下调作用。以上结果提示不同亚型PPARs对胎盘CRH的表达和分泌呈不同的调节作用。5、收集正常妊娠和PIH患者胎盘组织,检测CBS、CSE mRNA和蛋白的表达、H₂S的生成速率,结果显示PIH患者胎盘CBS和CSE mRNA及蛋白表达、H₂S的生成速率等较正常者显著减少。统计学相关性分析结果表明胎盘组织PPARγ表达与CSE和CBS的表达呈负相关,而PPARγ表达与CSE的表达呈正相关,提示胎盘PPARs可能参与维持胎盘H₂S合成酶表达的调节过程。结论:1、1)不同PPARs对11β-HSD2mRNA表达的调节作用不需要通过合成新蛋白、不影响11β-HSD2 mRNA的稳定性,而是直接调节11β-HSD2基因的转录,对胎盘11β-HSD2的表达和酶活性呈现不同甚至相反的调节作用;PPARγ可能通过与RXR形成异源二聚体促进胎盘11β-HSD2的表达和活性,而PPARγ对胎盘11?β-HSD2的调节过程可能不需要RXR的参与。2)胎盘组织PPARγ与11β-HSD2之间、PPARγ与11β-HSD2之间的表达具有相关性,提示PPARs可能参与胎盘11β-HSD2表达的精细调节过程,即不同PPARs亚型的表达水平和活性之间的平衡可能是胎盘维持11β-HSD2正常范围的重要影响因素;2、1)胎盘表达CSE和CBS,并能生成H₂S;H₂S抑制胎盘细胞CRH的合成和分泌,提示胎盘局部H₂S可能通过调控CRH的水平而参与胎盘内分泌功能的调节;2)不同亚型PPARs对胎盘H₂S合成酶CBS、CSE的表达具有不同甚至相反的调节作用;3)PIH患者胎盘与正常妊娠胎盘相比,CBS、CSE mRNA和蛋白表达、H₂S生成速率均下降,提示H₂S在PIH患者的生成减少;胎盘PPARs的表达与CSE和CBS的表达具有相关性,提示不同亚型PPARs参与胎盘H₂S合成酶正常表达的调节过程;4)不同亚型PPARs对胎盘CRH具有不同的调节作用,但此作用是PPARs的直接作用还是通过影响胎盘H₂S合成酶表达的间接作用还有待进一步研究。更多还原

【Abstract】 Glococorticoids are essential for normal fetal organ growth and maturation. However, excessive glucocorticoid exposure in uterus leads to intrauterine growth restriction （IUGR） and pregnancy-induced hypertension （PIH）. The placental enzyme 11β- hydroxysteroid dehydrogenase type 2 （11β-HSD2） is believed to play a key role in protecting the fetus from exposure to high levels of maternal glucocorticoid by converting maternal cortisol to its inactive metabolite, cortisone. Indeed, placental 11β-HSD2 activity is correlated with birth weight and is attenuated in pregnancies complicated with IUGR. Thus, the precise control of placental 11β-HSD2 expression and activity appears to be critical to normal fetal development. However, the understanding of placental 11β-HSD2 regulation is incomplete.Peroxisome proliferatoractivated receptors （PPARs） are members of the nuclear receptor superfamily. Three distinct PPAR subtypes have been identified, namely, PPAR?, PPAR??（also known as PPAR?） and PPAR?, with unique tissue distributions and physiological functions. Three PPAR subtypes have been found to express in the human placenta, which function in this organ is unclear. Recently PPARs-null mice exhibit placental defects and reduced birth weight, indicating a pivotal role for this nuclear receptor in placental function and fetal development. We hypothesized that this nuclear receptor may regulate human placental function in part by targeting 11β-HSD2. In the first part of present study, we conducted experiments to determine the modulation and mechanism of PPARs on 11β-HSD2 using cultured human placental trophoblast cells as a model system.The pharmacological, physiological and pathological roles of gasotransmitters nitric oxide （NO） and carbon monoxide （CO） have been extensively researched in the reproductive system. Hydrogen sulphide （H₂S） is another gasotransmitter that has many parallels with NO and CO. However there are few reports to date on the production and function of H₂S in reproductive tissues. H₂S is endogenously produced from L-cysteine by two pyridoxal 5’ phosphate-dependent enzymes cystathionineβ-synthase （CBS） and cystathionineγ-lyase （CSE）. Recently the presence of CBS and CSE enzymes was demonstrated in human intrauterine tissues including placenta. However the role and regulation of H₂S in the placenta are unclear. In the second part, we conducted experiments to observe expression level of H₂S synthetases in normal placenta and PIH placenta; to determine if PPARs modulate the expression of CBS and CSE in human placental trophoblast. As we known, Corticotropin-Releasing Hormone （CRH） takes a vital part in the progression of pregnancy and initiation of labor in human. However, the mechanism of CRH regulation is fully uncovered. So further experiment was conducted to observe whether H₂S and PPARs influence the synthesis and release of CRH in human placenta.Main results and conclusions are as follows:1 . Regulation and Mechanism of 11β-HSD2 Expression in Human Placenta Syncytiotrophoblasts by PPARs1) Treatment of placental cells with PPARγagonist GW7647 caused a decrease in the mRNA and protein expression as well as the activity of 11β-HSD2. These effects could be blocked by PPARγantagonist GW6471. PPARγagonist rosiglitazone dose-dependently stimulated the expression and activity of 11β-HSD2. PPARγantagonist GW9662 reversed the effects of rosiglitazone.2) Placental Cells were treated with PPARs agonists in the absence and presence of CHX, a protein synthesis inhibitor. It was found that GW7647 and rosiglitazone were equally effective in modulating 11β-HSD2 mRNA in the absence and presence of CHX, indicating that de novo protein synthesis was not required.3) RXR ligand 9-cis-RA alone could increase the expression of 11β-HSD2 in the placental cells. And 9-cis-RA potentiated the effect induced by PPAR? agonist. Transfected the placental cells with RXRαsiRNA abolished the effect by rosiglitazone, whereas the effect of GW7647 was not influenced. The results suggested that the modulation of PPARγnot PPAR? depended on RXRα.4) To elucidate the molecular mechanisms by which PPARs regulated 11β-HSD2 mRNA, cells were treated with DRB, an inhibitor of mRNA synthesis. The results showed that GW7647 and rosiglitazone did not alter the half-life of 11β-HSD2 mRNA. 5) GW7647 repressed whereas rosiglitazone stimulated the promoter activity of 11β-HSD2 gene. GW6471 and GW9662 could reverse the effects of GW7647 and rosiglitazone respectively. The results suggested that GW7647 and rosiglitazone could regulate genetic transcription of 11β-HSD2.6) Western blot hybridization and real-time PCR showed that the expression of 11β-HSD2, ?and PPARγwere significantly lower in placentas from women with PIH compared with those with normal pregnant, while the expression of PPARγand PPARγwas opposite. Moreover, Statistical analyses showed that there is a negative correlation between the expression of 11β-HSD2 and PPARγ, whereas a positive correlation existed between the expression of 11β-HSD2 and PPARγin human placenta. Results suggested that PPARs involve in maintain of 11β-HSD2 expression in placenta.2.Regulation of Synthetases Responsible for H₂S Production in Human Placenta Syncytiotrophoblasts by PPARs.1) Both H₂S synthetases, CBS and CSE, express in human placenta showed by immunochemistry.2) Treatment the placental cells with L-cysteine or sodium hydrosulfide, CRH mRNA expression and CRH IR concentration in media and cell lysis of cells decreased. The effect of L-cycsteine was blocked in the presence of CSE antagonist PAG or CBS antagonist AOAA. Meanwhile D-cysteine had no effect on CRH mRNA and IR concentration. The results suggested that H₂S regulate CRH synthesis and secretion in placenta.3) PPARγagonist GW7647 and PPARβagonist GW0742 significantly down-regulated the mRNA and protein expression of CBS and CSE in placental trophoblast cells, while PPARγagonist rosiglitazone stimulated the expression of CBS and CSE. The above effects of PPARs were reversed by PPARs subtypes antagonists repectively.4) Treatment the placental cells with PPARαand PPARβagonists, CRH mRNA expression and IR concentration in media and cell lysis of cells increased. The effects of PPARγand PPARγwere reversed by PPARγand PPARγantagonist respectively. Meanwhile PPARγhad down-regulation on CRH mRNA expression and IR concentration.5) The mRNA and protein expression of CBS and CSE were significantly decreased in PIH placental biopsies compared to those of normal samples. Statistical analyses showed that there is a negative correlation between PPARγand CBS, PPARγand CSE, while a positive correlation existed between CSE and PPARγin human placenta. To define the real-time kinetics of H₂S production by placenta, we used miniaturized H₂S micro-respiration sensor to measure its production. Results showed that the level of H₂S production in PIH placenta significantly decreased compared to normal placenta. It suggested that H₂S maybe involve in the pathological progress of PIH.In summary, the above results demonstrated:These data suggested that PPARγand PPARγdifferentially modulate the expression and activity of 11β-HSD2 in human trophoblast cells, these effects are mediated primarily at transcriptional level. Moreover, the results suggested that PPARs regulate the expression of CBS and CSE in human placenta, which effects were different. Furthermore, the above effects could modulate the production of H₂S in placenta, which further effect the synthesis and secrete of CRH in placenta. In fact the results showed that both H2S and PPARs subtypes coulud modulate the synthsis and secretion of CRH in placnta.Also the results showed that there is a correlation between the expression of 11β-HSD2 and PPARs, H2S Synthetases and PPARs in human placenta. Thus, the present study indicates that PPARs may modulate the human placental function, and, consequently, fetal development.更多还原