人绒毛膜促性腺激素促进胎盘糖皮质激素屏障11β-HSD2表达的机制研究
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摘要
适量的糖皮质激素对胎儿器官的发育和成熟起着重要作用,据此原理,临床至今仍然将糖皮质激素作为促进早产儿肺成熟的首选药物,这对于提高早产儿的存活率、降低新生儿湿肺的发生率等非常重要。但如果妊娠期胎儿暴露于过量的糖皮质激素则对胎儿不利,不仅可以导致胎儿宫内发育迟缓,而且增加胎儿成年后患高血压、心脑血管疾病、糖尿病等代谢性疾病的几率。目前,这些由孕期暴露于不良因素导致的成年疾病已引起国际学术界的高度重视,被称之为“胎儿起源的成人疾病”(fetal origins of adult disease, FOAD)。因此,妊娠期适量控制胎儿体内糖皮质激素的量对优生优育有重要意义。妊娠期母体肾上腺分泌的糖皮质激素量随孕期逐渐增加,而胎儿肾上腺主要以合成脱氢表雄酮(DHEA)为主,直到孕晚期才能合成糖皮质激素。孕期胎儿体内的糖皮质激素浓度仅为母体侧的十分之一,且主要来源于胎儿自身合成的糖皮质激素,因此胎儿需要一道糖皮质激素保护屏障。以往实验已经充分表明,担任糖皮质激素保护屏障的是一种糖皮质激素的代谢酶,即11β-羟基类固醇脱氢酶Ⅱ型(11β-HSD2),它主要在胎盘的合体滋养层细胞表达,其作用是将具有生物活性的糖皮质激素皮质醇(cortisol, F)脱氢氧化为没有生物活性的17-羟-11-脱氢皮质酮(可的松,cortisone, E)。在其作用下,母体来源的皮质醇主要以17-羟-11-脱氢皮质酮的形式进入胎儿体内,从而保证胎儿在低糖皮质激素的环境中生长发育。动物实验发现,抑制胎盘11β-HSD2的功能将导致胎盘糖皮质激素屏障的削弱,胎儿不仅出现宫内发育迟缓,还出现下丘脑-垂体-肾上腺轴的变化,且成年后出现高血压、血糖升高等代谢性疾病。这不仅进一步印证了11β-HSD2的胎盘糖皮质激素屏障功能,而且也提示了研究胎盘11β-HSD2表达调控的重要临床意义。
我们以往的研究发现,一氧化氮通过cGMP途径下调胎盘11β-HSD2的表达和活性,而forskolin激活cAMP/PKA途径则上调胎盘11β-HSD2的表达和活性。cAMP/PKA途径对于维持胎盘糖皮质激素屏障非常重要,但通过cAMP/PKA途径上调胎盘cAMP/PKA表达的胎盘内源性激素至今不清楚。因此我们试图在胎盘分泌的激素中筛选一种通过cAMP/PKA途径上调胎盘11β-HSD2的激素。在所有胎盘分泌的以cAMP为第二信使的激素中,我们发现绒毛膜促性腺激素(hCG)的分泌特点与胎盘11β-HSD2似乎有某种联系。HCG是孕期维持妊娠的重要激素,它属于糖蛋白家族,这一家族还包括促甲状腺激素释放激素(TSH)、黄体生成素(LH),由α和β两个不同的亚基组成,其中α亚基为糖蛋白家族共有亚基,而β亚基为特异性亚基。介导hCG作用的受体对LH也有很高的亲和力,因此称之为hCG/LH受体。hCG/LH受体的第二信使为cAMP,近期研究表明hCG不仅在孕早期对卵巢、胎盘有作用,而且在孕中晚期对胎盘、胎儿均有作用。HCG能促进胎儿肾上腺合成硫酸脱氢表雄激素(DHEAS),以提供给胎盘进一步合成雌激素。HCG能维持孕晚期子宫静息状态,从而起到保胎作用。HCG还能促进胎盘血管生成,促进胎盘植入,对孕期子宫动脉血管扩张也具有重要作用。妊娠6周后,合成hCG的细胞由细胞滋养层细胞转变为合体滋养层细胞,并于妊娠第8-10周时其合成的hCG的量达到顶峰。而此时,胎盘合体滋养层的糖皮质激素屏障也已建立,其转化皮质醇为可的松的能力也在第8-12周时达到高峰,提示胎盘合体滋养层分泌的hCG量与其表达的11β-HSD2量可能存在密切关系。无独有偶,胎盘合成和表达的hCG与11β-HSD2之间的关系也同样表现在妊娠晚期：虽然妊娠晚期胎盘仍具有相当强的分泌hCG和转化皮质醇为17-羟-11-脱氢皮质酮的能力,但于足月分娩前2周胎盘分泌的hCG量出现一定程度下降,而此时胎盘11β-HSD2的表达量也出现一定程度下降,这进一步提示胎盘分泌hCG的量与11β-HSD2的表达量存在一定的相关性。因此我们推测hCG很可能就是通过cAMP/PKA途径促进胎盘11β-HSD2表达的内源性激素。在上述推测被证明成立后,另一个问题随即出现。有文献报道糖皮质激素能促进胎盘11b-HSD2表达,而以往研究还发现糖皮质激素能促进孕早期和孕晚期胎盘合体滋养层细胞hCG的合成和释放,那么hCG是否参与介导糖皮质激素对胎盘11β-HSD2的上调作用呢?我们同样对这一问题进行了探讨。
我们采用改良Kliman法获得胎盘滋养层细胞,通过体外细胞培养,对胎盘合体滋养层细胞进行研究。我们的研究结果表明：hCG是通过cAMP/PKA途径促进胎盘11β-HSD2表达的内源性激素。而且hCG介导了糖皮质激素对11β-HSD2的上调作用。我们的实验首次证明了hCG对胎盘糖皮质激素屏障的作用,丰富了胎盘糖皮质激素屏障的调节机制,加深了我们对胎盘的理解,为临床应用hCG保胎以及优生优育提供理论基础。
研究内容
我们的实验主要对孕晚期胎盘合体滋养层细胞进行体外培养,探讨胎盘合体滋养层细胞糖皮质激素屏障11β-HSD2的调节机制。我们主要研究通过cAMP/PKA途径上调胎盘合体滋养层细胞11β-HSD2的内源性激素是什么?通过对胎盘分泌的众多激素的分析及实验我们确定hCG能通过cAMP/PKA途径上调胎盘合体滋养层细胞11β-HSD2。以往实验发现糖皮质激素本身能上调胎盘合体滋养层细胞11β-HSD2的表达,我们推测hCG可能介导糖皮质激素对胎盘合体滋养层细胞11β-HSD2的上调作用。通过实验我们进一步证实了hCG能介导糖皮质激素对胎盘合体滋养层细胞11β-HSD2的上调作用。材料与方法
1、孕晚期胎盘合体滋养层细胞体外培养方法。取正常妊娠(不合并妊娠期糖尿病、妊娠期高血压等),足月妊娠(孕38周-40周),行择期剖宫产的胎盘组织(50g左右),用改良Kliman酶消化法体外分离胎盘细胞滋养层细胞并进行体外培养。体外培养24-48小时后,胎盘细胞滋养层细胞自发融合形成合体滋养层细胞,即我们的研究对象。
2、对体外培养的胎盘合体滋养层细胞进行加药处理。培养48h后,用PBS清洗细胞,换培养液,然后向合体滋养层细胞加入cAMP/PKA通路激动剂forskolin.阻断剂H89、外源性hCG、hCG-Ab、F等实验药物后,培养12h或24h。随后对细胞进行相应处理。
3、通过QT-RT-PCR、Western blotting方法测定细胞内某一基因的mRNA水平及蛋白水平表达。
4、通过显微镜观察及苏木精-伊红染色方法(HE染色)对体外分离细胞的成分、合体滋养层细胞融合情况进行观察。
5、通过酶联免疫发光法对细胞培养液中的β-hCG浓度进行定量测定。
6、cAMP-GloTM ssay试剂盒测定细胞内cAMP的水平。
7、通过质粒转染胎盘细胞滋养层细胞、测定转染质粒的报告基因的荧光活性来分析外加因素对11β-HSD2启动子水平的影响。
实验结果
1、我们通过酶联免疫发光法测定细胞培养液中细胞分泌的β-hCG浓度,发现培养48小时后细胞分泌的hCG浓度即达到高峰。HCG是胎盘滋养层细胞分化的重要指标,hCG分泌浓度达到高峰后即认为胎盘合体滋养层细胞分化完全。因此证明我们分离的胎盘细胞滋养层细胞经过48小时的体外培养后完全分化融合为胎盘合体滋养层细胞,可以作为研究对象。我们通过苏木精-伊红染色方法(HE染色法)以及显微镜观察培养细胞的形态观察到我们培养的合体滋养层细胞合体形成很好。我们分离的合体滋养层细胞纯度高、活性好。
2、通过QT-RT-PCR、Western blotting方法证明hCG能通过cAMP/PKA途径上调胎盘合体滋养层细胞11β-HSD2的mRNA水平和蛋白水平表达。通过报告基因方法我们发现forskolin、H89、hCG、hCG-Ab能相应影响11β-HSD2启动子的转录水平。hCG-Ab能减少合体滋养层细胞内cAMP水平,从而使11β-HSD2表达量下降。
3、通过QT-RT-PCR、Western blotting方法证明糖皮质激素能上调胎盘合体滋养层细胞11β-HSD2的mRNA水平和蛋白水平表达,酶联免疫发光法测定细胞培养液中β-hCG浓度表明糖皮质激素能促进胎盘合体滋养层细胞hCG的表达和释放。通过QT-RT-PCR、Western blotting方法证明糖皮质激素对胎盘合体滋养层细胞11β-HSD2上调作用至少有一部分是通过hCG实现的。
结论：
hCG是通过cAMP/PKA途径促进胎盘合体滋养层细胞表达11β-HSD2的重要内分泌和旁分泌激素,而且hCG介导了糖皮质激素对胎盘合体滋养层细胞表达11β-HSD2的上调作用。
Proper glucocorticoid exposure in utero is vital for normal fetal organ maturation, but excess glucocorticoids are detrimental to fetal growth and can even predispose the individuals to the high risk of having certain diseases in adulthood, such as hypertension, diabetes and strokes. It has been proposed that the fetus is protected from 10 times higher maternal glucocorticoid levels by the placental enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which converts biologically active cortisol to inactive cortisone requiring NAD as its cofactor. Importantly,11β-HSD2, with a Michaelis constant (Km) value in the nanomolar range, is suited well to regulate the passage of maternal glucocorticoids into the fetal circulation. It was demonstrated that at term all the cortisone in the fetus was of maternal origin, suggesting that placental 11β-HSD2 was acting as an effective glucocorticoid barrier at term. Alterations in placental 11β-HSD2 expression and activity are known to be associated with reduced fetal growth in placentae of human pregnancies complicated by intrauterine growth restriction (IUGR) and adult hypertension. This placental glucocorticoid barrier was functional not only at term, but also at the early and mid gestational ages.
The expression of 11β-HSD2 was localized to the syncytiotrophoblast, which is the component of the human placenta involved not only in fetal maternal exchanges, but also in secretion of pregnancy-specific hormones such as human chorionic gonadotropin (hCG). Human CG is a glycoprotein hormone composed of two nonidentical subunits,αandβ, that are associated nonconvalently. The role of hCG in the maintenance of pregnancy is well known for the most extensively studied function of stimulating progesterone synthesis as well as the tocolytic action of promoting uterine quiescence. Studies have also shown its involvement in the regulation of implantation, trophoblastic hormonal secretion and trophoblast differentiation. It is well established that the stimulation of hCG receptors activates adenylate cyclase, resulting in the production of cyclic adenosine monophosphate (cAMP) and subsequent protein kinase A (PKA) activation. Intact hCG has been detected in maternal blood as early as around the 8th day of gestation. After the 6th week of gestation, the major site of hCG synthesis is switched from cytotrophoblast to syncytiotrophoblast with the maximal concentrations of hCG in maternal plasma attained at 8 th to 10 th weeks gestation, a period when the glucocorticoid barrier is beginning to take shape. In addition, in parallel with a significant decrease in serum hCG level 2-3 weeks before the spontaneous start of labor, there was a decline in placental 11β-HSD2 activity as well. The coincidence of hCG secretion and 11β-HSD2 expression in the placenta strongly indicates a close relationship between these two events. Although accumulating evidence indicates that activation of cAMP/PKA pathway plays an important role in up-regulating 11β-HSD2 expression in placental syncytiotrophoblasts, the endogenous hormone produced by the placenta utilizing this pathway to maintain 11β-HSD2 expression remains largely unknown. Previous studies have found that injections of hCG could stimulate 11β-HSD2 expression in the fish testis and rat uterus, suggesting it is very likely that hCG could be one of the endogenous hormones utilizing cAMP/PKA pathway to maintain 11β-HSD2 expression in human placenta.
As the substrate for 11β-HSD2, glucocorticoids have been shown to increase placental 11β-HSD2 expression in vitro, which would represent an important safeguard mechanism by which the fetus be protected from detrimental exposure to elevated levels of maternal glucocorticoids. Since glucocorticoids have been reported to stimulate hCG secretion from the placenta, we speculate that the effect of glucocorticoids on placental 11β-HSD2 expression could be mediated at least in part through the stimulation of hCG secretion.
In this study, we examined the role of hCG in maintaining placental 11β-HSD2 expression and in mediating the induction of placental 11β-HSD2 expression by cortisol in cultured human placental syncytiotrophoblasts.
Materials and Methods
1、Placenta Trophoblast Cell Culture. Human placentae were obtained from uncomplicated normal term pregnancies after elective caesarean section. Placental trophoblast cells were prepared using a modification of the method of Kliman. After being cultured for 48h placental trophoblast cells fused to form syncytiotrophoblasts spontaneously which were used for study.
2、Treatment of Syncytiotrophoblasts in Culture. The cells were treated with forskolin (the adenylate cyclase stimulator, 100uM), H89 (PKA inhibitor, 20uM), hCG, hCG antibody, cortisol respectively for 12h or 24h.
3、The total RNA and protein were extracted from the cells treated as above for analysis with realtime-PCR and Western blotting for the purpose of quantitative analysis of the target genes.
4、Using hematoxylin and eosin stain (HE stain) and microscope to determine the maximal formation of syncytiotrophoblasts and the purity of syncytiotrophoblasts.
5、Using chemiluminescence immunoassay (CLIA) for the quantitative analysis of p-hCG in the culturing medium.
6、Using cAMP-GloTM Assay kit to measure intracellular cAMP concentration.
7、Transfection of Placental Trophoblasts with pGL3 Plasmid Carrying 11β-HSD2 Promoter-driven Reporter Gene followed by measurement of Promoter Activity.
Results
1、Microscopic morphological examination of the cells showed that maximal fusion of the cells was found to occur at 48 h after plating. Measurement of the hCG level in the culture medium showed that the secretion of hCG by the cells reached a plateau at 48 h after plating suggesting maximal formation of syncytiotrophoblasts occurred at this time point. Thus this incubation time was chosen to allow maximal formation of syncytiotrophoblasts before experimentation in this study.
2、Human Chorionic Gonadotropin Up-regulates 11β-HSD2 Expression via cAMP/PKA Pathway in Human Placenta Syncytiotrophoblasts. Measurement with qRT-PCR revealed that treatment of the syncytiotrophoblasts with adenylate cyclase stimulator forskolin (100uM) for 24 h increased 11β-HSD2 mRNA level significantly, while inhibition of PKA with H89 (20uM) decreased 11β-HSD2 mRNA level significantly. Neutralization of hCG secreted by the syncytiotrophoblasts with different concentrations of hCG antibody (1:80,1:125,1:250) for 24 h reduced 11β-HSD2 mRNA level in a concentration-dependent manner. Blocking the endogenous hCG with its antibody (1:100) also decreased 11β-HSD2 protein level. In the mean time, treatment of the cells with hCG antibody (1:100) for 4 h decreased cAMP level in the syncytiotrophoblasts. In contrast to the results of hCG antibody treatment, treatment of the syncytiotrophoblasts with exogenous hCG (10 IU/ml) for 12 h increased both 11β-HSD2 and protein levels, which were blocked by H89 (20uM). Forskolin (100uM) treatment of the syncytiotrophoblasts transfected with pGL3 basic plasmid carrying 330bp 11β-HSD2 promoter for 24 h increased the promoter activity significantly, while both H89 (20uM,24h) and hCG antibody (1:100) treatment of the syncytiotrophoblasts for 24 h significantly decreased 11 p-HSD2 promoter activity.
3、Involvement of hCG in the Up-regulation of 11β-HSD2 Expression by Cortisol in Human Placenta Syncytiotrophoblasts. Treatment of the syncytiotrophoblasts with cortisol (0.01-luM) for 24 h increased 11β-HSD2 mRNA level in a concentration dependent manner. Protein synthesis inhibitor CHX (10uM) completely abolished the induction of 11β-HSD2 mRNA expression by cortisol (1uM), suggesting the induction by cortisol requires de novo protein synthesis. Cortisol (1uM) treatment of the syncytiotrophoblasts for 24h increased both hCG and subunit mRNA levels and hCG subunit level in the culture medium. Furthermore, the induction of 11β-HSD2 mRNA and protein expression by cortisol (1uM,24h) could be blocked by either hCG antibody (1:100) or H89 (20uM).
Conclusion
We demonstrated for the first time that hCG is an important paracrine or autocrine hormone maintaining 11β-HSD2 expression and the up-regulation of 11β-HSD2 expression by cortisol may be mediated in part by hCG in the syncytiotrophoblasts.
我们以往的研究发现,一氧化氮通过cGMP途径下调胎盘11β-HSD2的表达和活性,而forskolin激活cAMP/PKA途径则上调胎盘11β-HSD2的表达和活性。cAMP/PKA途径对于维持胎盘糖皮质激素屏障非常重要,但通过cAMP/PKA途径上调胎盘cAMP/PKA表达的胎盘内源性激素至今不清楚。因此我们试图在胎盘分泌的激素中筛选一种通过cAMP/PKA途径上调胎盘11β-HSD2的激素。在所有胎盘分泌的以cAMP为第二信使的激素中,我们发现绒毛膜促性腺激素(hCG)的分泌特点与胎盘11β-HSD2似乎有某种联系。HCG是孕期维持妊娠的重要激素,它属于糖蛋白家族,这一家族还包括促甲状腺激素释放激素(TSH)、黄体生成素(LH),由α和β两个不同的亚基组成,其中α亚基为糖蛋白家族共有亚基,而β亚基为特异性亚基。介导hCG作用的受体对LH也有很高的亲和力,因此称之为hCG/LH受体。hCG/LH受体的第二信使为cAMP,近期研究表明hCG不仅在孕早期对卵巢、胎盘有作用,而且在孕中晚期对胎盘、胎儿均有作用。HCG能促进胎儿肾上腺合成硫酸脱氢表雄激素(DHEAS),以提供给胎盘进一步合成雌激素。HCG能维持孕晚期子宫静息状态,从而起到保胎作用。HCG还能促进胎盘血管生成,促进胎盘植入,对孕期子宫动脉血管扩张也具有重要作用。妊娠6周后,合成hCG的细胞由细胞滋养层细胞转变为合体滋养层细胞,并于妊娠第8-10周时其合成的hCG的量达到顶峰。而此时,胎盘合体滋养层的糖皮质激素屏障也已建立,其转化皮质醇为可的松的能力也在第8-12周时达到高峰,提示胎盘合体滋养层分泌的hCG量与其表达的11β-HSD2量可能存在密切关系。无独有偶,胎盘合成和表达的hCG与11β-HSD2之间的关系也同样表现在妊娠晚期：虽然妊娠晚期胎盘仍具有相当强的分泌hCG和转化皮质醇为17-羟-11-脱氢皮质酮的能力,但于足月分娩前2周胎盘分泌的hCG量出现一定程度下降,而此时胎盘11β-HSD2的表达量也出现一定程度下降,这进一步提示胎盘分泌hCG的量与11β-HSD2的表达量存在一定的相关性。因此我们推测hCG很可能就是通过cAMP/PKA途径促进胎盘11β-HSD2表达的内源性激素。在上述推测被证明成立后,另一个问题随即出现。有文献报道糖皮质激素能促进胎盘11b-HSD2表达,而以往研究还发现糖皮质激素能促进孕早期和孕晚期胎盘合体滋养层细胞hCG的合成和释放,那么hCG是否参与介导糖皮质激素对胎盘11β-HSD2的上调作用呢?我们同样对这一问题进行了探讨。
我们采用改良Kliman法获得胎盘滋养层细胞,通过体外细胞培养,对胎盘合体滋养层细胞进行研究。我们的研究结果表明：hCG是通过cAMP/PKA途径促进胎盘11β-HSD2表达的内源性激素。而且hCG介导了糖皮质激素对11β-HSD2的上调作用。我们的实验首次证明了hCG对胎盘糖皮质激素屏障的作用,丰富了胎盘糖皮质激素屏障的调节机制,加深了我们对胎盘的理解,为临床应用hCG保胎以及优生优育提供理论基础。
研究内容
我们的实验主要对孕晚期胎盘合体滋养层细胞进行体外培养,探讨胎盘合体滋养层细胞糖皮质激素屏障11β-HSD2的调节机制。我们主要研究通过cAMP/PKA途径上调胎盘合体滋养层细胞11β-HSD2的内源性激素是什么?通过对胎盘分泌的众多激素的分析及实验我们确定hCG能通过cAMP/PKA途径上调胎盘合体滋养层细胞11β-HSD2。以往实验发现糖皮质激素本身能上调胎盘合体滋养层细胞11β-HSD2的表达,我们推测hCG可能介导糖皮质激素对胎盘合体滋养层细胞11β-HSD2的上调作用。通过实验我们进一步证实了hCG能介导糖皮质激素对胎盘合体滋养层细胞11β-HSD2的上调作用。材料与方法
1、孕晚期胎盘合体滋养层细胞体外培养方法。取正常妊娠(不合并妊娠期糖尿病、妊娠期高血压等),足月妊娠(孕38周-40周),行择期剖宫产的胎盘组织(50g左右),用改良Kliman酶消化法体外分离胎盘细胞滋养层细胞并进行体外培养。体外培养24-48小时后,胎盘细胞滋养层细胞自发融合形成合体滋养层细胞,即我们的研究对象。
2、对体外培养的胎盘合体滋养层细胞进行加药处理。培养48h后,用PBS清洗细胞,换培养液,然后向合体滋养层细胞加入cAMP/PKA通路激动剂forskolin.阻断剂H89、外源性hCG、hCG-Ab、F等实验药物后,培养12h或24h。随后对细胞进行相应处理。
3、通过QT-RT-PCR、Western blotting方法测定细胞内某一基因的mRNA水平及蛋白水平表达。
4、通过显微镜观察及苏木精-伊红染色方法(HE染色)对体外分离细胞的成分、合体滋养层细胞融合情况进行观察。
5、通过酶联免疫发光法对细胞培养液中的β-hCG浓度进行定量测定。
6、cAMP-GloTM ssay试剂盒测定细胞内cAMP的水平。
7、通过质粒转染胎盘细胞滋养层细胞、测定转染质粒的报告基因的荧光活性来分析外加因素对11β-HSD2启动子水平的影响。
实验结果
1、我们通过酶联免疫发光法测定细胞培养液中细胞分泌的β-hCG浓度,发现培养48小时后细胞分泌的hCG浓度即达到高峰。HCG是胎盘滋养层细胞分化的重要指标,hCG分泌浓度达到高峰后即认为胎盘合体滋养层细胞分化完全。因此证明我们分离的胎盘细胞滋养层细胞经过48小时的体外培养后完全分化融合为胎盘合体滋养层细胞,可以作为研究对象。我们通过苏木精-伊红染色方法(HE染色法)以及显微镜观察培养细胞的形态观察到我们培养的合体滋养层细胞合体形成很好。我们分离的合体滋养层细胞纯度高、活性好。
2、通过QT-RT-PCR、Western blotting方法证明hCG能通过cAMP/PKA途径上调胎盘合体滋养层细胞11β-HSD2的mRNA水平和蛋白水平表达。通过报告基因方法我们发现forskolin、H89、hCG、hCG-Ab能相应影响11β-HSD2启动子的转录水平。hCG-Ab能减少合体滋养层细胞内cAMP水平,从而使11β-HSD2表达量下降。
3、通过QT-RT-PCR、Western blotting方法证明糖皮质激素能上调胎盘合体滋养层细胞11β-HSD2的mRNA水平和蛋白水平表达,酶联免疫发光法测定细胞培养液中β-hCG浓度表明糖皮质激素能促进胎盘合体滋养层细胞hCG的表达和释放。通过QT-RT-PCR、Western blotting方法证明糖皮质激素对胎盘合体滋养层细胞11β-HSD2上调作用至少有一部分是通过hCG实现的。
结论：
hCG是通过cAMP/PKA途径促进胎盘合体滋养层细胞表达11β-HSD2的重要内分泌和旁分泌激素,而且hCG介导了糖皮质激素对胎盘合体滋养层细胞表达11β-HSD2的上调作用。
Proper glucocorticoid exposure in utero is vital for normal fetal organ maturation, but excess glucocorticoids are detrimental to fetal growth and can even predispose the individuals to the high risk of having certain diseases in adulthood, such as hypertension, diabetes and strokes. It has been proposed that the fetus is protected from 10 times higher maternal glucocorticoid levels by the placental enzyme 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which converts biologically active cortisol to inactive cortisone requiring NAD as its cofactor. Importantly,11β-HSD2, with a Michaelis constant (Km) value in the nanomolar range, is suited well to regulate the passage of maternal glucocorticoids into the fetal circulation. It was demonstrated that at term all the cortisone in the fetus was of maternal origin, suggesting that placental 11β-HSD2 was acting as an effective glucocorticoid barrier at term. Alterations in placental 11β-HSD2 expression and activity are known to be associated with reduced fetal growth in placentae of human pregnancies complicated by intrauterine growth restriction (IUGR) and adult hypertension. This placental glucocorticoid barrier was functional not only at term, but also at the early and mid gestational ages.
The expression of 11β-HSD2 was localized to the syncytiotrophoblast, which is the component of the human placenta involved not only in fetal maternal exchanges, but also in secretion of pregnancy-specific hormones such as human chorionic gonadotropin (hCG). Human CG is a glycoprotein hormone composed of two nonidentical subunits,αandβ, that are associated nonconvalently. The role of hCG in the maintenance of pregnancy is well known for the most extensively studied function of stimulating progesterone synthesis as well as the tocolytic action of promoting uterine quiescence. Studies have also shown its involvement in the regulation of implantation, trophoblastic hormonal secretion and trophoblast differentiation. It is well established that the stimulation of hCG receptors activates adenylate cyclase, resulting in the production of cyclic adenosine monophosphate (cAMP) and subsequent protein kinase A (PKA) activation. Intact hCG has been detected in maternal blood as early as around the 8th day of gestation. After the 6th week of gestation, the major site of hCG synthesis is switched from cytotrophoblast to syncytiotrophoblast with the maximal concentrations of hCG in maternal plasma attained at 8 th to 10 th weeks gestation, a period when the glucocorticoid barrier is beginning to take shape. In addition, in parallel with a significant decrease in serum hCG level 2-3 weeks before the spontaneous start of labor, there was a decline in placental 11β-HSD2 activity as well. The coincidence of hCG secretion and 11β-HSD2 expression in the placenta strongly indicates a close relationship between these two events. Although accumulating evidence indicates that activation of cAMP/PKA pathway plays an important role in up-regulating 11β-HSD2 expression in placental syncytiotrophoblasts, the endogenous hormone produced by the placenta utilizing this pathway to maintain 11β-HSD2 expression remains largely unknown. Previous studies have found that injections of hCG could stimulate 11β-HSD2 expression in the fish testis and rat uterus, suggesting it is very likely that hCG could be one of the endogenous hormones utilizing cAMP/PKA pathway to maintain 11β-HSD2 expression in human placenta.
As the substrate for 11β-HSD2, glucocorticoids have been shown to increase placental 11β-HSD2 expression in vitro, which would represent an important safeguard mechanism by which the fetus be protected from detrimental exposure to elevated levels of maternal glucocorticoids. Since glucocorticoids have been reported to stimulate hCG secretion from the placenta, we speculate that the effect of glucocorticoids on placental 11β-HSD2 expression could be mediated at least in part through the stimulation of hCG secretion.
In this study, we examined the role of hCG in maintaining placental 11β-HSD2 expression and in mediating the induction of placental 11β-HSD2 expression by cortisol in cultured human placental syncytiotrophoblasts.
Materials and Methods
1、Placenta Trophoblast Cell Culture. Human placentae were obtained from uncomplicated normal term pregnancies after elective caesarean section. Placental trophoblast cells were prepared using a modification of the method of Kliman. After being cultured for 48h placental trophoblast cells fused to form syncytiotrophoblasts spontaneously which were used for study.
2、Treatment of Syncytiotrophoblasts in Culture. The cells were treated with forskolin (the adenylate cyclase stimulator, 100uM), H89 (PKA inhibitor, 20uM), hCG, hCG antibody, cortisol respectively for 12h or 24h.
3、The total RNA and protein were extracted from the cells treated as above for analysis with realtime-PCR and Western blotting for the purpose of quantitative analysis of the target genes.
4、Using hematoxylin and eosin stain (HE stain) and microscope to determine the maximal formation of syncytiotrophoblasts and the purity of syncytiotrophoblasts.
5、Using chemiluminescence immunoassay (CLIA) for the quantitative analysis of p-hCG in the culturing medium.
6、Using cAMP-GloTM Assay kit to measure intracellular cAMP concentration.
7、Transfection of Placental Trophoblasts with pGL3 Plasmid Carrying 11β-HSD2 Promoter-driven Reporter Gene followed by measurement of Promoter Activity.
Results
1、Microscopic morphological examination of the cells showed that maximal fusion of the cells was found to occur at 48 h after plating. Measurement of the hCG level in the culture medium showed that the secretion of hCG by the cells reached a plateau at 48 h after plating suggesting maximal formation of syncytiotrophoblasts occurred at this time point. Thus this incubation time was chosen to allow maximal formation of syncytiotrophoblasts before experimentation in this study.
2、Human Chorionic Gonadotropin Up-regulates 11β-HSD2 Expression via cAMP/PKA Pathway in Human Placenta Syncytiotrophoblasts. Measurement with qRT-PCR revealed that treatment of the syncytiotrophoblasts with adenylate cyclase stimulator forskolin (100uM) for 24 h increased 11β-HSD2 mRNA level significantly, while inhibition of PKA with H89 (20uM) decreased 11β-HSD2 mRNA level significantly. Neutralization of hCG secreted by the syncytiotrophoblasts with different concentrations of hCG antibody (1:80,1:125,1:250) for 24 h reduced 11β-HSD2 mRNA level in a concentration-dependent manner. Blocking the endogenous hCG with its antibody (1:100) also decreased 11β-HSD2 protein level. In the mean time, treatment of the cells with hCG antibody (1:100) for 4 h decreased cAMP level in the syncytiotrophoblasts. In contrast to the results of hCG antibody treatment, treatment of the syncytiotrophoblasts with exogenous hCG (10 IU/ml) for 12 h increased both 11β-HSD2 and protein levels, which were blocked by H89 (20uM). Forskolin (100uM) treatment of the syncytiotrophoblasts transfected with pGL3 basic plasmid carrying 330bp 11β-HSD2 promoter for 24 h increased the promoter activity significantly, while both H89 (20uM,24h) and hCG antibody (1:100) treatment of the syncytiotrophoblasts for 24 h significantly decreased 11 p-HSD2 promoter activity.
3、Involvement of hCG in the Up-regulation of 11β-HSD2 Expression by Cortisol in Human Placenta Syncytiotrophoblasts. Treatment of the syncytiotrophoblasts with cortisol (0.01-luM) for 24 h increased 11β-HSD2 mRNA level in a concentration dependent manner. Protein synthesis inhibitor CHX (10uM) completely abolished the induction of 11β-HSD2 mRNA expression by cortisol (1uM), suggesting the induction by cortisol requires de novo protein synthesis. Cortisol (1uM) treatment of the syncytiotrophoblasts for 24h increased both hCG and subunit mRNA levels and hCG subunit level in the culture medium. Furthermore, the induction of 11β-HSD2 mRNA and protein expression by cortisol (1uM,24h) could be blocked by either hCG antibody (1:100) or H89 (20uM).
Conclusion
We demonstrated for the first time that hCG is an important paracrine or autocrine hormone maintaining 11β-HSD2 expression and the up-regulation of 11β-HSD2 expression by cortisol may be mediated in part by hCG in the syncytiotrophoblasts.
引文
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2、D. J. P. Barker, C.Osmond, P. D/Weight in infancy and death from ischaemic heart disease/The Lancet. Volume 334, Issue 8663,9 September 1989, Pages 577-580.
3、Evensen KAI, et al./Effects of preterm birth and fetal growth retardation on cardiovascular risk factors in young adulthood./Early Hum Dev (2008), doi:10.1016/j. earlhumdev.2008.10.008
4、孙刚《胎盘内分泌基础与临床》。
5、K. Sun, K. Yang/Glucocorticoid Actions and Metabolism in Pregnancy: Implications for Placental Function and Fetal Cardiovascular Activity./ Placenta (1998),19,353-360.
6、Jonathan R. Seckl/Glucocorticoids, feto-placental 11β-hydroxysteroid dehydrogenase type 2 and the early life origins of adult disease/S teroids 62:89-94,1997.
7、Mulder et al.,/Antenatal corticosteroid therapy:short-term effects on fetal behaviour and haemodynamics,/Seminars in Fetal & Neonatal Medicine (2008), doi:10.1016/j. siny.2008.10.003
8、DAVID J. P. BARKER, MD, PHD, FRCP/Maternal Nutrition, Fetal Nutrition, and Disease in Later Life/Nutrition Vol.13, No.9,1997.
9、Simitzis, P. E., et al./Influence of maternal undernutrition on the behaviour of juvenile lambs. Appl. Anim. Behav. Sci. (2008), doi:10.1016/j. applanim.2008.09.007.
10、Francesca Mastorci, Massimo Vicentini./Long-term effects of prenatal stress:Changes in adult cardiovascular regulation and sensitivity to stress./Neuroscience & Biobehavioral Reviews. Volume 33, Issue 2, February 2009, Pages 191-203
11、H. Nunez et al./Fetal undernutrition induces overexpression of CRH mRNA and CRH protein in hypothalamus and increases CRH and corticosterone in plasma during postnatal life in the rat./Neuroscience Letters 448 (2008) 115-119.
12、F. Mastorci et al./Long-term effects of prenatal stress:Changes in adult cardiovascular regulation and sensitivity to stress/Neuroscience and Biobehavioral Reviews 33 (2009) 191-203.
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1、David J. P. Barker/Fetal programming of coronary heart disease./TRENDS in Endocrinology & Metabolism Vol.13 No.9 November 2002.
2、D. J. P. Barker, C.Osmond, P. D/Weight in infancy and death from ischaemic heart disease/The Lancet. Volume 334, Issue 8663,9 September 1989, Pages 577-580.
3、Evensen KAI, et al./Effects of preterm birth and fetal growth retardation on cardiovascular risk factors in young adulthood./Early Hum Dev (2008), doi:10.1016/j. earlhumdev.2008.10.008
4、孙刚《胎盘内分泌基础与临床》。
5、K. Sun, K. Yang/Glucocorticoid Actions and Metabolism in Pregnancy: Implications for Placental Function and Fetal Cardiovascular Activity./ Placenta (1998),19,353-360.
6、Jonathan R. Seckl/Glucocorticoids, feto-placental 11β-hydroxysteroid dehydrogenase type 2 and the early life origins of adult disease/S teroids 62:89-94,1997.
7、Mulder et al.,/Antenatal corticosteroid therapy:short-term effects on fetal behaviour and haemodynamics,/Seminars in Fetal & Neonatal Medicine (2008), doi:10.1016/j. siny.2008.10.003
8、DAVID J. P. BARKER, MD, PHD, FRCP/Maternal Nutrition, Fetal Nutrition, and Disease in Later Life/Nutrition Vol.13, No.9,1997.
9、Simitzis, P. E., et al./Influence of maternal undernutrition on the behaviour of juvenile lambs. Appl. Anim. Behav. Sci. (2008), doi:10.1016/j. applanim.2008.09.007.
10、Francesca Mastorci, Massimo Vicentini./Long-term effects of prenatal stress:Changes in adult cardiovascular regulation and sensitivity to stress./Neuroscience & Biobehavioral Reviews. Volume 33, Issue 2, February 2009, Pages 191-203
11、H. Nunez et al./Fetal undernutrition induces overexpression of CRH mRNA and CRH protein in hypothalamus and increases CRH and corticosterone in plasma during postnatal life in the rat./Neuroscience Letters 448 (2008) 115-119.
12、F. Mastorci et al./Long-term effects of prenatal stress:Changes in adult cardiovascular regulation and sensitivity to stress/Neuroscience and Biobehavioral Reviews 33 (2009) 191-203.