【作者】 杨臻；

【导师】 孙刚；

【作者基本信息】 复旦大学 ， 生理与生物物理学， 2010， 博士

【摘要】 胎儿器官成熟特别是肺成熟与分娩发动是一个协同进行的过程,而这一过程依赖于妊娠晚期内源性糖皮质激素的增加。由于人类胎儿肾上腺的特殊结构和胎盘糖皮质激素屏障的存在,妊娠晚期胎儿组织11β-羟基类固醇脱氢酶Ⅰ型(11β-Hydroxysteroid dehydrogenase type 1,11β-HSD1)对17-羟-11-脱氢皮质酮的活化作用可能成为胎儿体内糖皮质激素的重要来源。目前已有充分证据表明,大多数动物胎儿肺能够表达11β-HSD1,而且11β-HSD1-/-小鼠肺成熟出现障碍,提不11β-HSD1在胎儿肺成熟中的关键作用,但人胎儿组织特别是肺是否表达11β-HSD1目前还不明了。以往研究发现,人胎膜表达的11β-HSD1也为胎儿器官成熟提供重要的糖皮质激素来源,糖皮质激素和促炎性细胞因子作为抑炎和促炎的两种因子却都可以诱导胎膜11β-HSD1的表达,但其分子机制目前尚未阐明。本课题采用免疫组织化学、常规PCR、Western blotting、实时荧光定量PCR、酶活性测定、启动子克隆与活性测定、功能蛋白过表达、染色质免疫沉淀(ChIP)和凝胶电泳迁移实验(EMSA)等技术,首先研究了人胎儿肺组织是否表达11β-HSD1,然后分别以人胎儿肺成纤维细胞株(HFL-1)和原代培养人羊膜成纤维细胞为研究模型,研究了促炎性细胞因子(白细胞介素-1β)和糖皮质激素(皮质醇)诱导11β-HSD1表达的分子机制。1.常规PCR和免疫组织化学染色结果表明,妊娠中期(孕4个月)和晚期(孕8个月)人胎肺组织中都能够检测到11β-HSD1 mRNA,但只在妊娠晚期胎肺组织中检测到11β-HSD1的分布,包括小支气管和细支气管的上皮细胞和小支气管间质细胞。此外,在HFL-1细胞中也检测到了11β-HSD1mRNA和蛋白,而且用Western blotting和酶活性测定的方法证实了上述结果。此结果不但提示胎儿肺组织至少在妊娠晚期可以表达11β-HSD1蛋白,而且也提示HFL-1细胞是进一步研究胎儿肺11β-HSD1表达调控的良好实验模型。2.以HFL-1细胞和原代培养的人羊膜成纤维细胞为研究模型,用实时荧光定量PCR、Western blotting和酶活性测定等方法研究发现,白细胞介素-1β与皮质醇增加11β-HSD1 mRNA和蛋白水平的现象同时存在于HFL-1细胞和原代培养的人羊膜成纤维细胞中。3.实时荧光定量PCR测定结果表明,白细胞介素-1β与皮质醇对人羊膜成纤维细胞和HFL-1细胞11β-HSD1 mRNA表达的诱导作用能够被mRNA转录抑制剂DRB和蛋白质合成抑制剂CHX阻断,提示白细胞介素-1β与皮质醇增加11β-HSD1mRNA水平的作用发生在转录水平并需要蛋白质合成的参与。用表达拮抗C/EBP蛋白的质粒(CMV-500-A-C/EBP)转染HFL-1细胞和人羊膜成纤维细胞发现,白细胞介素-1β与皮质醇诱导11β-HSD1 mRNA表达作用均可以被不同程度阻断,提示在上述两种细胞中C/EBPs家族成员均参与了白细胞介素-1β与皮质醇诱导11β-HSD1 mRNA表达作用。4.我们以HFL-1细胞为模型进一步研究了白细胞介素-1β诱导11β-HSD1 mRNA表达的分子机制。结果表明：(1)NF-kB转录激活抑制剂5HPP-33不能阻断白细胞介素-1β对HFL-1细胞11β-HSD1 mRNA表达的诱导作用,但能够阻断白细胞介素-1β对炎症介质前列腺素合成酶COX-2 mRNA的诱导作用,提示白细胞介素-1β对11β-HSD1 mRNA表达的促进作用与此经典的炎症通路无关。(2)将11β-HSD1启动子克隆并构建于报告基因质粒,转染HFL-1细胞后测定其启动子活性发现,白细胞介素-1β能够促进11β-HSD1启动子活性增加,此作用可以被CCAAT序列突变所阻断。(3)Western blotting分析结果表明,HFL-1细胞中主要表达C/EBPβ而几乎不表达C/EBPα,白细胞介素-1β强烈诱导C/EBPβ的表达。(4)进一步用ChIP和EMSA技术研究发现,白细胞介素-1β促进C/EBPβ与11β-HSD1启动子CCAAT序列的结合。(5)转染表达C/EBPβ质粒(pMSV-C/EBPβ)和表达拮抗C/EBP蛋白的质粒(CMV-500-A-C/EBP)分别可以模拟和阻断白细胞介素-1β对11β-HSD1表达的诱导作用。以上结果提示,白细胞介素-1β主要通过C/EBPβ诱导HFL-1细胞11β-HSD1的表达。5.我们以原代培养的人羊膜成纤维细胞为模型进一步研究了皮质醇诱导11β-HSD1 mRNA表达的分子机制。结果表明：(1)皮质醇对11β-HSD1 mRNA表达的诱导作用能够被糖皮质激素受体拮抗剂RU486阻断,提示皮质醇诱导11β-HSD1表达的作用需要糖皮质激素受体的参与。(2)将11β-HSD1启动子-报告基因质粒转染细胞后测定其启动子活性发现,皮质醇能够促进人羊膜成纤维细胞11β-HSD1启动子活性增加,此作用可以分别被糖皮质激素反应元件(GRE)和CCAAT序列突变所削弱。(3)Western blotting分析结果表明,人羊膜成纤维细胞中主要表达C/EBPa而少量表达C/EBPp,皮质醇对人羊膜成纤维细胞C/EBPa表达的诱导作用比较明显。(4)进一步用ChIP和EMSA技术研究发现,皮质醇促进GRα和C/EBPa分别与11β-HSD1启动子的GRE和CCAAT序列的结合。(5)转染CMV-500-A-C/EBP质粒可以削弱皮质醇对11β-HSD1表达的诱导作用。以上结果提示,糖皮质激素受体和C/EBPa都参与了皮质醇对人羊膜成纤维细胞11β-HSD1表达的诱导作用。结论本课题研究结果表明,妊娠晚期人胎肺组织中表达11β-HSD1;白细胞介素-1β与皮质醇诱导11β-HSD1基因表达的现象同时存在于HFL-1细胞和人羊膜成纤维细胞。皮质醇主要通过促进糖皮质激素受体GRα和C/EBPa分别与11β-HSD1基因启动子糖皮质激素反应元件和C/EBP结合位点的结合诱导人羊膜成纤维细胞11β-HSD1基因表达；白细胞介素-1β主要通过促进C/EBPp与11β-HSD1基因启动子上C/EBP结合位点的结合诱导HFL-1细胞11β-HSD1基因表达。C/EBPs可能是皮质醇与白介素-1β诱导11β-HSD1基因表达的共同分子节点。更多还原

【Abstract】 Context and Aims:Synchronization of fetal maturation, especially the lung maturation, and initiation of parturition is crucial for the survival of the newborn. Accumulating evidences suggest that increased glucocorticoid level towards the end of gestation triggers both of the events. Due to the unique structure of the fetal adrenal and the existence of placental glucocorticoid barrier, the regeneration of cortisol from cortisone by 11β-hydroxysteroid dehydrogenase type 1 (11(3-HSD1) in the fetal tissue plays a significant role in elevating the local level of glucocorticoids within the tissues. Substantial evidence indicated the presence of 11β-HSD1 in most of the animal fetal lung tissues. Furthermore, 11β-HSD1-/- mice exhibit impaired fetal lung maturation, suggesting the crucial role of 11β-HSD1 in fetal lung maturation. However, the expression of 11β-HSD1 in human fetal lung remains controversial. In addition to the glucocorticoid regeneration by the fetal organ per se, glucocorticoids derived from 11β-HSD1 in the fetal membranes is believed to be another important extra-adrenal source of fetal cortisol in late gestation. Of interest, up-regulation of 11β-HSD1 by proinflammatory cytokines and anti-inflammatory glucocorticoids has been reported in the fetal membranes; however, the molecular mechanism underlying this intriguing up-regulation of 11β-HSD1 by proinflammatory cytokines and glucocorticoids remains largely unknown. In this study, we investigated the expression of 11β-HSD1 in human fetal lung tissues and examined whether the up-regulation of 11β-HSD1 by proinflammatory cytokines and glucocorticoids also holds true in the fetal lung cells. Based on these findings, we investigated the molecular mechanisms underlying the induction of 11 P-HSD1 by Interlukin-1β(IL-1β) and cortisol in human fetal lung fibroblast cell line (HFL-1) and primary human amnion fibroblasts respectively. Elucidation of these mechanisms may help us to understand better the process of human fetal lung maturation.Methods and Results:1. Regular PCR and immunohistochemistry revealed that 11β-HSD1 mRNA was detectable in the fetal lung tissues both at 8 and 4 months of gestation, but 11β-HSD1 protein was only detected in the fetal lung tissue at 8 months of gestation. Positive immunostaining for 11β-HSD1 was found in the epithelium lining the small bronchus, bronchiole and in the fibroblast-enriched mesenchymal tissue of the small bronchus. The detection of 11β-HSD1 mRNA, protein and enzyme activity in HFL-1 cells lends further support for the expression of 11β-HSD1 in human fetal lung. Meanwhile, our findings also indicate that HFL-1 cell is a good model for the further study of the regulation of 11β-HSD1 expression.2. By using real-time PCR, Western blotting and enzyme activity assay, we demonstrated that IL-1βand cortisol induced 11β-HSD1 expression in HFL-1 cells as well as in human amnion fibroblasts.3.Further studies with real-time PCR illustrated that the induction of 11β-HSD1 in HFL-1 cells and human amnion fibroblasts by both IL-1βand cortisol could be blocked by the messenger RNA transcription inhibitor DRB and the protein synthesis inhibitor CHX, suggesting that the induction is dependent on ongoing transcription and synthesis of at least one other protein. Of interest, the induction of 117β-HSD1 in HFL-1 cells and human amnion fibroblasts by both IL-1βand cortisol could also be blocked by transfection of CMV-500-A-C/EBP which expresses dominant negative C/EBP, suggesting that C/EBPs might be involved in the induction of 11β-HSD1 by both IL-1βand cortisol in both HFL-1 cells and human amnion fibroblasts.4. We further studied molecular mechanisms of the induction of 11β-HSD1 by IL-1βin HFL-1 cells. We found that:(1) NF-kB transactivation inhibitor 5HPP-33 could block IL-1β-induced COX-2 expression but not 11β-HSD1 expression, suggesting a unique pathway underlying the induction of 11β-HSD1 by IL-1β, which differs from the classical pro-inflammatory signaling pathway. (2) Reporter gene assay demonstrated that IL-1βstimulated 11β-HSD1 promoter activity, which was attenuated by mutagenesis of a C/EBP binding site in the promoter. (3) Western blotting results illustrated that HFL-1 cells expressed barely detectable C/EBPa but high level of C/EBPβthat could be induced dramatically by IL-1β. (4) C/EBPβwas found bound to the putative C/EBP binding site in 11β-HSD1 promoter upon IL-1βstimulation of HFL-1 cells with ChIP and EMSA. (5) Transfection of pMSV-C/EBPβplasmid over-expressing C/EBPβor CMV-500-A-C/EBP could mimic or antagonize the induction of 11β-HSD1 by IL-1β. The above results indicate that the induction of 11β-HSDl gene expression by IL-1βdepends on the activation of C/EBPβbinding to the CCAAT box in the promoter in HFL-1 cells.5. The molecular mechanisms underlying the induction of 11β-HSD1 by cortisol were further investigated in primary human amnion fibroblasts. Results showed that:(1) The induction of 11β-HSD1 by cortisol could be blocked by GR antagonist RU486, suggesting the induction is a GR-dependent process. (2) Report gene assay demonstrated that the induction of 11β-HSD1 promoter activity by cortisol was attenuated by mutagenesis of a glucocorticoid response element (GRE) and a CCAAT consensus sequence in the promoter, respectively. (3) Western blotting illustrated that human amnion fibroblasts expressed low C/EBPβbut high C/EBPαlevel that can be increased by cortisol. (4) ChIP and EMSA demonstrated that both GRαand C/EBPαcould bind the GRE and C/EBP binding site upon cortisol stimulation of human amnion fibroblasts (5) The induction of 11β-HSD1 by cortisol could be blocked by transfection of CMV-500-A-C/EBP. All these results lead to the conclusions that induction of 11β-HSD1 gene expression by cortisol involves both activation of GRαand C/EBPαbinding to the promoter in human amnion fibroblasts.Conclusions:11β-HSD1 is expressed in human fetal lung tissue at least at later gestation. The induction of 11β-HSD1 by IL-1βand cortisol holds true in HFL-1 cells as well as in primary human amnion fibroblasts. The induction of 11β-HSD1 gene expression by IL-1βdepends on the activation of C/EBPβbinding to the CCAAT box in the promoter in HFL-1 cells, while the induction of 11β-HSD1 gene expression by cortisol involves both activation of GRαand C/EBPαbinding to the promoter in human amnion fibroblasts. Based upon all these findings, we propose that C/EBPs be the key transcription factors involved in the induction of 11β-HSD1 by both IL-1βand cortisol.更多还原