糖皮质激素对人绒毛膜滋养层细胞11β-HSD1的调节

目的：研究糖皮质激素对11β-羟基类固醇脱氢酶I型(11β-hydroxysteroid dehydiogenase type1,11β-HSD1)还原酶活性和mRNA表达的调节。方法：利用原代培养的人类绒毛膜滋养层细胞,运用免疫细胞化学染色方法、放射性酶活性测定及Northem印迹杂交技术,结合图像分析技术检测11β-HSD1 mRNA的表达量。结果：11β-HSD1和糖皮质激素受体(glucocorticoid receptor,GR)免疫活性样物质共存于原代培养的人绒毛膜滋养层细胞中,人工合成的糖皮质激素——地塞米松对绒毛膜滋养层细胞11β-HSD1还原酶活性和mRNA表达具有诱导作用,此诱导作用可以被GR阻断剂RU486所阻断。结论：糖皮质激素与GR结合后上调绒毛膜滋养层细胞11β-HSD1酶活性和mRNA表达。     

胎盘11β-HSD与病理妊娠的研究进展

病理妊娠包括胎儿生长受限、妊娠期高血压疾病、妊娠期糖尿病等,是严重威胁母婴健康的疾病。对病理妊娠机制的深入研究,有助于我们在临床上更好的诊治。妊娠期间胎盘分泌的糖皮质激素代谢酶11β-羟基类固醇脱氢酶(11β-HSD,11β-hydroxysteroid dehydrogenase)的异常表达被认为与病理妊娠有关。11β-HSD分为11β-HSD1和11β-HSD2两种亚型。胎盘11β-HSD2为避免胎儿暴露于过量的糖皮质激素水平提供了屏障作用,保证胎儿正常生长发育。胎盘11β-HSD2表达和活性降低与胎儿生长受限有关。胎盘11β-HSD2的活性在妊娠期高血压疾病患者中是降低的,妊娠期高血压疾病也可导致胎盘11β-HSD2活性的进一步降低。胎盘11β-HSD1表达增加与胰岛素抵抗有关,可能参与妊娠期糖尿病的发病。针对胎盘11β-HSD的活性调节将是病理妊娠治疗的重要措施之一。     

β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

目的：建立β淀粉样肽（Aβ1-40）诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法：用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子仪;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Western blot检测刺激后海马神经元内诱导型一氧化氮合酶（iNOS）和硝基酪氨酸（NT）的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3（caspase-3）活性来评价神经元的损伤程度。结果：终浓度为10μmol／L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24-72h,海马神经元较对照组形态有明显变化;经Western blot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论：小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。     

地塞米松致子代大鼠海马轴突发育损伤

为了探讨地塞米松对子代大鼠海马轴突的影响,建立了孕期地塞米松暴露（prenatal dexamethasone exposure, PDE）模型。Wistar大鼠于孕中晚期皮下注射地塞米松（0.2 mg·kg^-1·d^-1）,部分子代于孕20天（GD20）、出生后12周（PW12）处死取海马样本,检测海马糖皮质激素受体（glucocorticoid receptor, GR）活化指标以及轴突损伤指标。PDE子代胎鼠海马GR活化,GR、糖皮质激素调节激酶1（glucocorticoid-regulated kinase 1, SGK1）和FK506结合蛋白（FK506 binding protein 5, FKBP5）表达显著增加。轴突损伤指标包括生长相关蛋白43（growth associated protein-43,GAP43）、信号素3A（semaphorin 3A, SEMA3A）和集聚蛋白（agrin）表达明显升高。而PDE成年子代大鼠海马GR无明显活化,轴突损伤指标GAP43、SEMA3A和AGRIN表达明显升高。研究结果证实PDE通过活化胎海马GR引起轴突发育损伤,且轴突损伤可延续至出生后。     

吗啡对福尔马林引起大鼠海马内IL-2RβmRNA表达的影响

本实验采用原位杂交法观察足底注射福尔马林（Ｆｏｒ）痛敏对海马内白细胞介素２受体βｍＲＮＡ（ＩＬ－２ＲβｍＲＮＡ）生成的影响及其与吗啡、促肾上腺皮质激素（ＡＣＴＨ）的关系。结果表明：正常大鼠海马有ＩＬ－２ＲβｍＲＮＡ表达,集中分布于ＣＡ１－ＣＡ４区神经元、齿状回颗粒细胞。足底注射Ｆｏｒ后６ｈ双侧海马ＩＬ－２ＲβｍＲＮＡ表达均增加（Ｐ〈０．０５）,１２ｈ达高峰,２４ｈ仍高于正常。在６ｈ时,腹腔注射吗啡     

β-细辛醚对AD大鼠海马神经元蛋白质组图谱的影响

目的：探讨β-细辛醚对痴呆大鼠海马神经元蛋白质表达谱的影响。方法：SD大鼠随机分为正常时照组、模型组和β-细辛醚治疗组。模型组和β-细辛醚治疗组大鼠行脑立体定位注射术,模型组从大鼠右侧海马注入β淀粉样蛋白（Aβ1-40）5μg;治疗组：将Aβ1-40和β-细辛醚（0.12%）混合后注入大鼠右侧海马。抽提各组大鼠海马神经元组织蛋白质,行双向电泳,采用PDQuest-7.1.1分析软件解析各组之间有明显差异表达的蛋白质。结果：β-细辛醚治疗组α-烯醇化酶（α-enolase）、钙/钙调蛋白依赖蛋白激酶（Ca^2＋/Calmodulin-dependent protein kinase）的表达高于模型组,而尿激酶型纤溶酶原激活物（urokillasc plasminogen activator surface）、P53抑癌基因（P53 tumor suppressor）和未知蛋白（SSP6001）表达则低于模型组。结论：β-细辛醚可能通过上调或下调上述蛋白质的表达,参与促进大鼠海马受损神经元保护作用。     

低氧预处理对大鼠海巴神经元缺氧耐受性和IL—1β表达 …

目的：观察低氧预处理对大鼠海巴神经元缺氧耐受性和白细胞介素－１β（ＩＬ－１β）表达的影响。方法：取培养１２ｄ的两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（０．９Ｌ／ＬＮ２,０．１Ｌ／ＬＣＯ２）中培养２、４、８和１２ｈ。分别观察它们的形态变化和神经元存活数,并用抗ｒｈＩＬ－１β单克隆抗体进行免疫组织化学染色,观察缺氧对大鼠海马培养神经元ＩＬ－１β表达的影响。结果：经低氧预处理的海马神经     

在应激低反应期新生大鼠的海马和视交叉上核核受体-MR、GR表达的变化

目的搞清新生大鼠HPA轴改变在应激低反应时期（stress hyporesponsive period、SHRP））的核受体-MR（盐皮质激素受体）和GR（糖皮质激素受体）在海马和视交叉上核（SCN）的表达变化。从而进一步了解新生大鼠发育时间对MR和GR的影响。方法本研究应用免疫荧光技术研究了生后4d（PD4）和生后12d（PD12）的新生大鼠海马和SCN的MR、GR的表达。同时采用RIA方法测定了血清中皮质类固醇的浓度水平。结果RIA结果显示在应激低反应期给予刺激,血清皮质类固醇水平没有明显改变。在海马,PD12较PD4,MR-和GR-i mmunoreactivity在CA1区表达增强;在齿状回没有明显变化;在海马门（hilus）减弱。而在海马的CA3区和SCN中,PD4 GR-ir强烈表达,在PD12却下降。同时也发现在hilus细胞出现GR和nestin（巢蛋白）阳性表达的共存,但是却没有发现MR和nestin的共存。结论新生大鼠阶段,发育时间对MR和GR的影响因区域不同影响不同。GR和nestin（巢蛋白）的共存为探讨在新生大鼠阶段GR对神经干细胞的分化有否影响提供了实验资料。本研究也为进一步研究新生大鼠应激低反应状态下海马及SCN与低活性的HPA轴的关系提供了形态学依据。     

缺氧诱导体外培养大鼠海马神经元c-fos的表达及人重组白细胞介素-1β的影响

采用免疫组化方法,观察缺氧诱导体外培养大鼠海马神经元ｃ－ｆｏｓ的表达及人重组白细胞介素－１β（ｒｈＩＬ－１β）的影响。结果显示,缺氧后海马神经元中Ｆｏｓ染色阳性胞核的百分率随缺氧时间的延长而显著增加。图像分析的结果显示,缺氧后Ｆｏｓ染色阳性胞核的平均光密度亦随缺氧时间的延长而显著增加。经ｒｈＩＬ－１β孵育的神经元缺氧后Ｆｏｓ染色阳性胞核的百分率和Ｆｏｓ染色阳性胞核的平均光密度均明显低于对照组。本结果表明,缺氧能诱导体外培养海马神经元ｃ－ｆｏｓ表达,ｒｈＩＬ－１β能抑制缺氧神经元ｃ－ｆｏｓ表达。提示ｒｈＩＬ－１β对海马神经元缺氧损伤具有一定调控作用。     

地塞米松对大白鼠海马兴奋性神经元和抑制性神经元的影响

为了探讨糖皮质激素对海马兴奋性神经元和抑制性神经元的作用,本实验将地塞米松注入大白鼠侧脑室,２ｈ 后经Ｎｉｓｓｌ染色法、免疫组织化学方法和细胞计数法观察了海马谷氨酸免疫反应性（ＧｌｕＩＲ）神经元和γ氨基丁酸免疫反应性（ＧＡＢＡＩＲ）神经元的变化。结果显示：（１）ＣＡ１、ＣＡ３ 和ＳＧ区的ＧｌｕＩＲ神经元明显增多,特别是ＣＡ１ 区。经细胞计数统计分析表明,与对照组相比ＣＡ１ 有极显著性差异（Ｐ＜ ０００１）,ＣＡ３区有显著性差异（００１＜ Ｐ＜ ００５）,ＳＧ处无明显差异（Ｐ＞ ００５）。（２）与对照组相比,ＧＡＢＡＩＲ神经元无明显变化。结果表明,糖皮质激素有增加海马谷氨酸能神经元的作用。尽管γ氨基丁酸能神经元无明显变化,并不表明糖皮质激素对其无影响     

Intracrine induction of 11beta-hydroxysteroid dehydrogenase type 1 expression by glucocorticoid potentiates prostaglandin production in the human chorionic trophoblast

Glucocorticoids are involved in the modulation of the release of parturition hormones from the fetal membranes and placenta, where their actions are determined by the prereceptor glucocorticoid metabolizing enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). Two distinct isozymes of 11beta-HSD have been characterized. In the fetal membranes, 11beta-HSD1 is the predominate isozyme; it converts biologically inert 11-ketone glucocorticoid metabolites into active glucocorticoids. Sequence analysis of the cloned 11beta-HSD1 gene revealed a putative glucocorticoid response element in the promoter region. However, whether glucocorticoids modulate 11beta-HSD1 expression in the fetal membranes is unknown. In this study, 11beta-HSD1 and glucocorticoid receptor (GR) were coexpressed in the chorionic trophoblast. Radiometric conversion assay and Northern blot analysis revealed that both 11beta-HSD1 reductase activity and mRNA levels were increased by dexamethasone (1 microM, 0.1 microM) in the cultured chorionic trophoblast, and the effects were blocked by GR antagonist RU486 (1 microM). Prior induction of 11beta-HSD1 by dexamethasone potentiated the subsequent stimulation of prostaglandin H synthetase 2 expression and secretion of prostaglandin E(2) by cortisone in the chorionic trophoblast. There is colocalization of 11beta-HSD1 and GR in the chorionic trophoblast. By binding to GR, glucocorticoids induce the expression of 11beta-HSD1 by a possible intracrine mechanism, thereby amplifying the actions of glucocorticoids on prostaglandin production in the fetal membranes. This cascade of events initiated by glucocorticoids may play an important role in the positive feed-forward mechanisms of labor.     

Rapid spinogenesis of pyramidal neurons induced by activation of glucocorticoid receptors in adult male rat hippocampus

Modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. Dendritic spines are essential for memory storage processes. Here, we investigated the effect of dexamethasone (DEX), a specific agonist of glucocorticoid receptor (GR), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of Lucifer Yellow-injected spines in slices. The application of 100 nM DEX (stressful high concentration) induced rapid modulation of the density and morphology of dendritic spines in CA1 pyramidal neurons within 1h. The total spine density increased from 0.88 spines/microm (control) to 1.36 spines/microm (DEX-treated). DEX significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. Because the presence of 10 microM cycloheximide, an inhibitor of protein synthesis, did not suppress the DEX effect, these responses are probably non-genomic. Western immunoblot analysis demonstrated the localization of classical type GR in Triton-insoluble synaptosomal fractions (enriched in postsynaptic membranes) from hippocampal slices, suggesting a possible action site of DEX at spines.     

Spatial and temporal patterns of expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 messenger RNA and glucocorticoid receptor protein in the murine placenta and uterus during late pregnancy

To gain insight into the role of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes and actions of glucocorticoids in the murine placenta and uterus, the expression pattern of the mRNA for 11beta-HSD1 and 11beta-HSD2 and the glucocorticoid receptor (GR) protein were determined from Embryonic Day 12.5 (E12.5, term = E19) to E18.5 by in situ hybridization and immunohistochemistry, respectively. Consistent with its putative role in regulating the transplacental passage of maternal glucocorticoid to the fetus, 11beta-HSD2 mRNA was highly expressed in the labyrinthine zone (the major site of maternal/fetal exchange) at E12.5, and its level decreased dramatically at E16.5, when it became barely detectable. Remarkably, the silencing of 11beta-HSD2 gene expression coincided with the onset of 11beta-HSD1 gene expression in the labyrinth at E16.5 when moderate levels of 11beta-HSD1 mRNA were detected and maintained to E18.5. By contrast, neither 11beta-HSD1 mRNA nor 11beta-HSD2 mRNA were detected in any cell types within the basal zone from E12.5 to E18.5. Moreover, the expression of 11beta-HSD1 and 11beta-HSD2 in the decidua exhibited a high degree of cell specificity in that the mRNA for both 11beta-HSD1 and 11beta-HSD2 was detected in the decidua-stroma but not in the compact decidua. A distinct pattern was also observed within the endometrium where the mRNA for 11beta-HSD1 was expressed in the epithelium, whereas that for 11beta-HSD2 was confined strictly to the stroma. By comparison, the expression of GR in the placenta and uterus was ubiquitous and unremarkable throughout late pregnancy. In conclusion, the present study demonstrates for the first time remarkable spatial and temporal patterns of expression of 11beta-HSD1 and 11beta-HSD2 and GR in the murine placenta and uterus and highlights the intricate control of not only transplacental passage of maternal glucocorticoid to the fetus but also local glucocorticoid action during late pregnancy.     

11 Beta-hydroxysteroid dehydrogenase type 1 is induced in human monocytes upon differentiation to macrophages.

11beta-hydroxysteroid dehydrogenases (11beta-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11beta-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11beta-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11beta-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor gamma, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11beta-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11beta-HSD1 activity by up to 10-fold. IFN-gamma, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11beta-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11beta-HSD1 and low levels of 11beta-HSD2. The expression of 11beta-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11beta-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.     

Induction of human UGT1A1 by a complex of dexamethasone-GR dependent on proximal site and independent of PBREM

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role to conjugate bilirubin and prevent jaundice. There are two major elements for the induction of UGT1A1, such as PBREM (-3483/-3194), far from the promoter site, and HNF1 (-75/-63), near to the promoter site. In a previous report, we showed that the proximal HNF1 site is essential for the induction of UGT1A1 by glucocorticoid receptor (GR). In this report, we investigated the influence of PBREM on the induction of the UGT1A1 reporter gene by GR and PXR with dexamethasone (DEX). We confirmed that GR was transferred from cytosol into the nucleus in 15-30 min by DEX stimulation, but HNF1 was not. We constructed a reporter gene containing PBREM to compare the induction of the reporter gene without PBREM by DEX-GR. The results show that induction of the reporter gene with PBREM by DEX at 100 muM is the same level as the induction of the reporter gene without PBREM, although PBREM contains GRE. Co-transfection of hGR with the reporter gene did not show any influence of the induction of the reporter gene between the vector with and without PBREM. Meanwhile, by co-transfection of hPXR, the induction of the reporter gene with PBREM was significantly more than the induction of the reporter gene without PBREM at 100 muM DEX. This supports that hPXR induced UGT1A1 through PBREM by DEX. These results showed that PBREM has no relation with the induction by DEX-GR but the proximal site of UGT1A1 may function in stimulation by DEX-GR.     

Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat

Messenger RNAs coding for glucocorticoid (GR) and mineralocorticoid (MR) receptor proteins were localized to discrete subfields of the hippocampal formation by in situ hybridization histochemistry, using cRNA probes of approximately equivalent specific activity. Both GR and MR mRNAs were present in all subfields examined; GR mRNA was of greatest abundance in CA1, while MR mRNA was most densely labeled in CA3. In all subfields examined, MR mRNA was considerably more abundant than GR mRNA. Removal of circulating glucocorticoids by adrenalectomy precipitated an up-regulation of GR mRNA in subfields CA1-2 and the dentate gyrus, which was reversed by dexamethasone replacement. High doses of dexamethasone significantly down-regulated GR mRNA in CA3. In contrast, adrenalectomy produced significant up-regulation of MR mRNA only in subfield CA1-2. The data indicate that steroid receptor mRNAs are differentially distributed in hippocampus, and that sensitivity to steroids occurs within defined structural domains of the hippocampal formation.