17β-羟基类固醇脱氢酶

17β－羟基类固醇脱氢酶（17β HSD）为性激素合成中最后步骤的酶,催化氧化或还原反应,其作用是在受体前调节性激素的局部水平。各型17β HSD在体内有其特有的区域性分布,分布的特点与各自的功能有着密切的关系。17β HSD结构和功能的异常与一些疾病,如肿瘤、假两性等的发生有密切关系。     

大鼠海马神经元内11β-HSD1和GR的共存及其意义

本研究旨在探讨糖皮质激素代谢酶-11β-羟基类固醇脱氢酶Ⅰ型（11β-HSD1）和糖皮质激素受体（GR）在大鼠海马神经元内的共同分布及其意义。用免疫细胞化学方法研究显示,海马神经元内不仅存在11β-HSD1免疫反应物质,还存在GR免疫反应物质,而且11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLC）方法研究表明,地塞米松（DEX）可以促进11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLG）方法研究表明,地塞米松（DEX）可以促进11β-HSD1蛋白表达及其酶的活性,利用11β-HSD1基因启动子区序列构建的以CAT酶为报告基因的pBLCAT6质粒转染PC12细胞,证实DEX能够促进CAT酶的表达。以上糖皮质激素的作用均可为GR受体阻断剂RU38486所阻断,结果提示;糖皮质激素（GC）与GR结合后,可以作用于与其共存的11β-HSD1基因启动子区,使11β-HSD1表达增加,从而使更多的GC代谢产物转化为有活性的GC,此机制可能与保证GC在海马神经元内与亲和力较低的GR结合有关。     

3β-羟基类固醇脱氢酶基因的克隆表达及其性质研究

目的:构建3β-羟基类固醇脱氢酶(3β-HSD)异源表达系统,进一步探究其酶学性质。方法:通过分子生物学方法克隆来源于Mycobacterium neoaurum菌株的3β-羟基类固醇基因,构建重组质粒,运用HPLC方法检测酶反应体系的产物。结果:本实验构建了表达载体pet28a-hsd。优化诱导表达条件,发现3β-HSD异源表达的最适温度为16℃~25℃,37℃时蛋白表达为包涵体。此外,同一温度下不同IPTG浓度诱导的表达结果差异不大。利用纯化后的蛋白进行酶特性的研究,结果表明在3β-HSD酶反应体系中,30℃达到较高的酶活性;pH 7.5~9.0之间,酶活力较强,pH低于7.0时,酶活性明显下降;有机助溶剂DMSO对酶反应有抑制作用;Fe~(3+)和Cu~(2+)对酶反应有抑制作用。结论:本实验探究了分枝杆菌中3β-羟基类固醇脱氢酶的相关性质,为进一步研究该酶在类固醇代谢中的功能提供基础。     

脑内皮质激素受体及其受体前代谢酶的作用

中枢神经系统的发生、分化、发育成熟和退化中均有糖皮质激素的参与。糖皮质激素对神经系统的影响是在11β－羟基类固醇脱氢酶的调节下,通过与糖皮质激素受体和盐皮质激素受体结合,调节靶基因的转录而实现的。本文介绍了脑内糖皮质激素受体和11β－羟基类固醇脱氢酶的类型、分布、功能,以及二者在糖皮质激素发挥作用中的意义。     

人17β羟类固醇脱氢酶（17β—HSD）基因在家蚕系统中的表达（简报）

Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a crucial role in the synthesis of estrogens, but it also produces negative action of estrogens in promoting the growth of hormone-sensitive cancers, especially in breast and prostate. The high specific activity can be taken as an important signal for the diagnosis of cancers. Recombinant rAcBm-NPV/17 beta-HSD virus which contains the human 17 beta-HSD cDNA under the control of polyhedron gene promoter is generated by cotransfection of the BmN cells with the transfer plasmid pVL/17 beta-HSD and wild BmNPV genomic DNA. 17 beta-HSD is maximally expressed 72 h and 120 h post infection in BmN cells and the 5th instar silkworm larvae respectively. At those time interval, intracellular and hemolymphic enzymatic activity reach 0.12 U/mg and 0.15 U/mg of protein which produced total activity of 0.97 U/1.5 x 10(6) cells and 4.7 U/larva. The expressed quantities in female larvae are a little higher than that in male larvae. The present data shows that Silkworm/BmNPV expression system can express 3-5 times higher than that of the richest human placenta. It also indicates that there is an apparent band with a molecular mass of 35 kDa using SDS-PAGE method, the size of which is similar to that of the crude enzyme from placenta.     

人17β羟类固醇脱氢酶(17β-HSD)基因在家蚕系统中的表达(简报)

17β羟类固醇脱氢酶(17β-hydroxystero-id dehydrogenase,17β-HSD)能催化人体内类固醇性激素的形成和转化,例如它可以催化雌二醇与雌酮,雄烯二酮与睾酮之间的相互转化反应。人体内17β-HSD在胎盘组织含量最为丰富,催化17β-雌二醇等雌激素的生成,同时这些激素能够刺激乳腺瘤的增生。因此,如何抑制17β-HSD酶的过高活性,减少癌变发生的可能性,已成为目前这类癌症治疗的一个重要研究目标。目前,从胎盘组织中提取17β-HSD的方法,产量低,比活小,周期长,     

大鼠20α—羟类固醇脱氢酶在昆虫细胞中的表达

大鼠２０α羟类固醇脱氢酶（２０α－Ｈｙｄｒｏｘｙｓｔｅｒｏｉｄ ｄｅｈｙｄｒｏｇｅｎａｓｅ,２０αＨＳＤ）ｃＤＮＡ片段,被插入杆状病毒的转移载体ｐＢｌｕｅＢａｃⅢ,经野生型病毒ＤＮＡ的共转染,从被转染的昆虫细胞中获得重组病毒。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析,重组病毒感染细胞有２０αＨＳＤ基因表达。感染细胞裂解液的Ｗｅｓｔｅｒｎ印迹法分析,３７ｋＤ的蛋白带被２０αＨＳＤ抗体识别。体外酶活性测定发现,     

山羊胎儿肝脏3β—羟基甾体脱氢酶／Δ^4—Δ^5异构酶（3β—HSD）cDNA的克隆

3β－羟基甾体脱氢酶／Δ^4-Δ^5异构酶（3β－HSD）是哺乳动物体内广泛存在的一类参与甾体激素代谢的氧化还原酶,且具有异构酶活性。3β－HSD催化3β－羟基甾体的脱氢及随后的Δ^5-3-甾酮产物的异构化反应,以产生α,β－非饱和酮,3β－HSD在甾体激素代谢中起着重要作用。本文以胎羊肝为材料,首次获得了山羊胎肝的3β－HSD的cDNA,并进行了3β－HSD在肝脏组织的表达分析。参照牛,啮齿类的3β－HSD CDNA的高同源区设计引物,以2－3月雌性胎羊肝脏mRNA为模板,经RT－PCR获得416 bp cDNA片段（Fig.a),然后以其为探针筛选胎羊肝λgt10cDNA文库最后获得3－端缺失的3β－HSD cDNA克隆,并推导了其氨基酸序列（Fig.s)。同源分析表明山羊胎肝3β－HSD的氨基酸序列与牛卵巢,人I型3β－HSD的同源性分别为96％,78％和73％（Fig.3),提取雌性胎羊,雄性胎羊及母体羊肝脏的总RNA,同时做RT－PCR,表明3β－HSD在不同个体肝脏中的表达存在差异,雌性胎羊和母体孕羊肝脏中都有3β－HSD的表达,而在雄性胎羊肝脏中,则未检测到表达信号（Fig.4)。     

大鼠20α羟类固醇脱氢酶在大肠杆菌中的表达

利用谷胱甘肽Ｓ－转移酶融合基因表达系统,在鼠２０α羟类固醇脱氢酶在大肠杆菌中得以成功地表达,亲和层析和Ｔｈｒｏｍｂｉｎ消化,可从融合蛋白中回收和纯化重组２０αＨＳＤ。ＳＤＳ－ＰＡＧＥ,Ｗｅｓｔｅｒｎ印迹法和酶活性测定显示,重组２０α ＨＳＤ具有天然蛋白质相同分子量、相似的抗原性和酶催化活性,其中ＮＡＤＰ的Ｋｍ和Ｖｍａｘ分别为９．５μｍｏｌ／Ｌ、３３４ｎｍｏｌ／（ｍｉｎ．ｍｇ）,对底物２０α羟孕酮的     

11 beta-Hydroxysteroid dehydrogenase activity of the human placenta during pregnancy

A method for the quantitative estimation of 11 beta-hydroxysteroid dehydrogenase activity (11 beta-HSD; EC.1.1.146) in human placental homogenates is described. This method is based on the separation of cortisol and cortisone by high performance liquid chromatography after extraction from homogenates incubated in the presence of cortisol and NADP. 11 beta-HSD activity (pmol/g wet weight per min) averaged 900 +/- 150 (mean +/- SEM) at 10 +/- 2 weeks of gestation, 915 +/- 35 at 17 +/- 2 weeks and 790 +/- 42 at 40 +/- 2 weeks, thus supporting the view that the placenta is an effective barrier to materno-fetal cortisol transfer throughout gestation.     

11 beta-Hydroxysteroid dehydrogenase antisense affects vascular contractile response and glucocorticoid metabolism

Glucocorticoids (GC's) are metabolized in vascular tissue by two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). 11 beta-HSD2 is unidirectional and metabolizes GC's to their respective inactive 11-dehydro derivatives. 11 beta-HSD1 is bi-directional, also possessing reductase activity and thus the ability to regenerate active GC from the 11-dehydro derivatives. In vascular tissue, GC's amplify the pressor responses to catecholamines and angiotensin II and may down-regulate certain depressor systems such as nitric oxide and prostaglandins. We hypothesize that both 11 beta-HSD2 and 11 beta-HSD1 regulate GC levels in vascular tissue and are part of additional mechanisms that control vascular tone. We examined the effects of specific antisense oligomers to 11 beta-HSD2 and 11 beta-HSD1 on GC metabolism and contractile response to phenylephrine (PE) in rat aortic rings. In aortic rings incubated (24 h) with corticosterone (B) (10 nmol/l) and 11 beta-HSD2 antisense (3 micromol/l), the contractile response to graded concentrations of PE (PE: 10 nmol/l - 1 micromol/l) were significantly (P < 0.05) increased compared to rings incubated with B and 11 beta-HSD2 nonsense. 11 beta-HSD1 antisense oligomers also enhanced the ability of B to amplify the contractile response to PE. In addition, 11 beta-HSD2 and 11 beta-HSD1 antisense also decreased the metabolism of B to 11-dehydro-B. 11-Dehydro-B (100 nmol/l) also amplified the contractile response to PE in aortic rings (P < 0.01), most likely due to the generation of active corticosterone by 11 beta-HSD1-reductase; this effect was significantly attenuated by 11 beta-HSD1 antisense. 11 beta-HSD1 antisense also caused a marked decrease in the metabolism of 11-dehydro-B back to B by 11 beta-HSD1-reductase. These findings underscore the importance of 11 beta-HSD2 and 11 beta-HSD1 in regulating local concentrations of GC's in vascular tissue. They also indicate that decreased 11 beta-HSD2 activity may be a possible mechanism in hypertension and that 11 beta-HSD1-reductase may be a possible target for anti-hypertensive therapy.     

11beta-Hydroxysteroid dehydrogenase in the heart of normotensive and hypertensive rats

Corticosteroids have been shown to play a role in cardiac remodeling, with the possibility of a direct effect of overexpression of 11beta-hydroxysteroid dehydrogenase (11HSD) isoform 2 at the level of the cardiomyocytes. The aim of this study was to examine cardiac steroid metabolism in hypertensive rats with hearts that are hypertrophied and fibrotic and have structural alterations in the coronary circulation. To assess possible alterations of cardiac steroid metabolism the expression and activity of both isoforms of 11beta-hydroxysteroid dehydrogenase (11HSD) were studied in spontaneously hypertensive rats (SHR), their normotensive controls Wistar-Kyoto (WKY), and in Dahl salt-sensitive (DS) and salt-resistant rats (DR) kept on a low- or high-salt diet. Using real-time quantitative RT-PCR and enzyme activity assay we found strain-dependent differences in cardiac metabolism of glucocorticoids. In Dahl rats expression of 11HSD1 and 11HSD2 mRNA was lower in DS than in DR rats and was not influenced by dietary salt intake; 11HSD1 mRNA was expressed at higher level than 11HSD2 mRNA. NADP(+)-dependent cardiac 11HSD activity showed similar distribution as 11HSD1 mRNA-lower activity in DS than in DR rats and no effect of salt intake. In SHR and WKY strains 11HSD2 mRNA expression was significantly higher in WKY than in SHR but no differences were observed in 11HSD1 mRNA abundance and NADP(+)-dependent 11HSD activity. These results show that the heart is able to metabolize glucocorticoids and that this metabolism is strain-dependent but do not support the notion of association between cardiac hypertrophy and changes of 11HSD1 and 11HSD2 expression.     

11beta-Hydroxysteroid dehydrogenase expression and glucocorticoid synthesis are directed by a molecular switch during osteoblast differentiation

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in the prereceptor regulation of corticosteroids by locally converting cortisone into active cortisol. To investigate the impact of this mechanism on osteoblast development, we have characterized 11beta-HSD1 activity and regulation in a differentiating human osteoblast cell line (SV-HFO). Continuous treatment with the synthetic glucocorticoid dexamethasone induces differentiation of SV-HFO cells during 21 d of culture. Using this cell system, we showed an inverse relationship between 11beta-HSD1 activity and osteoblast differentiation. 11beta-HSD1 mRNA expression and activity were low and constant in differentiating osteoblasts. However, in the absence of differentiation (no dexamethasone), 11beta-HSD1 mRNA and activity increased strongly from d 12 of culture onward, with a peak around d 19. Promoter reporter studies provided evidence that specific regions of the 11beta-HSD1 gene are involved in this differentiation controlled regulation of the enzyme. Functional implication of these changes in 11beta-HSD1 is shown by the induction of osteoblast differentiation in the presence of cortisone. The current study demonstrates the presence of an intrinsic differentiation-driven molecular switch that controls expression and activity of 11beta-HSD1 and thereby cortisol production by human osteoblasts. This efficient mechanism by which osteoblasts generate cortisol in an autocrine fashion to ensure proper differentiation will help to understand the complex effects of cortisol on bone metabolism.     

3beta-Hydroxysteroid dehydrogenase activity in human fetal membranes.

A 3beta-hydroxysteroid dehydrogenase (3betaHSD) was demonstrated in term human fetal membranes (chorion and amnion) with both dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3beta-hydroxy-5-pregnen-20-one as substrates, and the subcellular distribution substrate and nucleotide specificity of the enzyme was studied. In both membranes the microsomal fraction (particles which sedimented at 105,000 g after 90 min) had the highest specific activity. The chorion was more active than the amnion but the enzyme in both tissues had similar substrate and nucleotide specificity. NAD was the preferred cofactor, and pregnenolone was a better substrate than dehydroepiandrosterone in the presence of NAD. However, with NADP as cofactor both steroids were equally good substrates. When the 3beta-hydroxysteroid dehydrogenase activity of chorion microsomes was compared with that of placental microsomes, the specific activities were found to be of the same order of magnitude, and the substrate, nucleotide specificity and steroid binding properties were almost identical.     

3 beta-Hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestinal bacteria

Ruminococcus sp. PO1-3 obtained from human intestinal flora is able to reduce dehydrocholate as well as 3-ketoglycyrrhetinate. From this bacterium dehydrocholate- and 3-ketoglycyrrhetinate-reducing activities were purified one thousand-fold together with 3-ketocholanate-reducing and 3-beta-hydroxyglycyrrhetinate (glycyrrhetic acid) oxidizing activities by means of Matrex Red A, Sephadex G-200 and Octyl-Sepharose column chromatography. The purified enzyme catalyzed the reduction of dehydrocholic acid to 3 beta-hydroxy-7,12-diketocholanic acid and of 3-ketocholanic acid to 3 beta-hydroxycholanic acid. Studies on substrate specificity revealed that the enzyme had absolute specificity for the beta-configuration of a hydroxyl group at the 3 position of bile acid and steroids having no double bond in the A/B ring. This enzyme was neither beta-hydroxysteroid dehydrogenase [EC 1.1.1.51] nor 3 beta-hydroxy-delta 5-steroid dehydrogenase [EC 1.1.1.145], but a novel type of enzyme, defined as 3 beta-hydroxysteroid dehydrogenase.     

17beta-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

17β-Hydroxysteroid dehydrogenase (17βHSD) type 13 is identified as a new lipid droplet-associated protein. 17βHSD type 13 has an N-terminal sequence similar to that of 17βHSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17βHSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17βHSD type 11, however, expression of 17βHSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor α and its ligand. Instead the expression level of 17βHSD type 13 in the receptor-null mice was increased several-fold. 17βHSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.