GRβ对肾小球系膜细胞糖皮质激素效应的影响

目的:构建糖皮质激素受体β(GRβ)不同表达水平的肾小球系膜细胞株,研究GRβ对肾小球系膜细胞糖皮质激素效应的影响.方法:利用逆转录病毒载体pLXSN将重组GRβ正义和反义基因转入肾小球系膜细胞,经细胞培养、逆转录聚合酶链反应、基因测序和蛋白质印迹分析鉴定所构建的细胞株,应用MTT法和流式细胞术检测细胞内不同表达水平的GRβ对地塞米松的细胞增殖抑制效应和细胞周期变化的影响.结果:构建的肾小球系膜细胞分别有正确的GRβ正义和反义基因整合,转染了GRβ正义基因的肾小球系膜细胞内GRβ蛋白质表达水平明显高于转染了GRβ反义基因者(109.74±10.63 vs.19.08±1.01,P＜0.05);地塞米松对GRβ高表达的肾小球系膜细胞的增殖抑制效率显著低于GRβ低表达的肾小球系膜细胞(18.47%±2.12% vs.60.33%±5.29%,P＜0.05).在地塞米松的抑制下,转染了正义GRβ基因的细胞中,其S期细胞降低程度和G1期细胞升高的程度均明显低于未转染的细胞.结论:肾小球系膜细胞内高表达的GRβ具有拮抗糖皮质激素效应的作用,细胞内GRβ表达水平是决定细胞对激素敏感或抵抗的重要因素.     

RNA干扰技术对人U937巨噬细胞株糖皮质激素受体表达及功能的抑制效应

目的利用载体介导的RNA干扰技术,建立糖皮质激素受体(GR)基因阻断的人U937巨噬细胞株模型.方法构建两个针对GR的RNAi重组表达载体,分别命名为pSilencer 3.1-GR1和pSilencer 3.1-GR2,经测序确认后,转染人U937巨噬细胞株.RT-PCR、Western blot分别检测糖皮质激素受体mRNA水平和蛋白水平表达变化;相对荧光素酶活性法检测地塞米松作用后GR转录激活功能的变化.结果经测序证实:成功构建两个针对GR的RNAi表达载体(pSilencer 3.1-GR1和pSilencer 3.1-GR2);转染pSilencer 3.1-GR2可明显降低细胞内GR mRNA的丰度及GR蛋白表达,细胞内GR转录激活功能明显降低;转染pSilencer 3.1-GR1与对照组相比无显著变化.结论成功地构建并筛选出一个高效且特异阻断GR表达及功能的RNAi质粒表达载体,为进一步研究糖皮质激素受体的功能提供新方法.     

RNA干扰技术抑制糖皮质激素受体在大鼠肺泡巨噬细胞表达及活性的研究

目的利用RNA干扰技术,建立糖皮质激素受体(glucocorticoid receptor,GR)基因阻断的大鼠肺泡巨噬细胞模型.方法构建2个针对GR的RNAi表达载体,分别命名为psilencer 3.1-GR1和psilencer 3.1-GR2,经测序确认后,转染大鼠肺泡巨噬细胞.RT-PCR、Western Blot分别检测GR mRNA水平和蛋白水平表达变化;相对荧光素酶活性法检测GR转录激活功能的变化.结果经测序证实:成功构建2个针对GR的RNAi表达载体(psilencer 3.1-GR1和2);转染psilencer 3.1-GR2可明显降低细胞内GR mRNA的丰度及GR蛋白表达,细胞GR转录激活活性明显降低;转染psilencer 3.1-GR1与对照组相比无显著变化.结论成功地构建并筛选出一个特异而高效地阻断GR表达及功能的质粒表达载体,为进一步研究糖皮质激素受体的功能提供新方法.     

脂多糖作用大鼠肺泡巨噬细胞糖皮质激素受体表达及活性变化

目的探讨脂多糖作用大鼠肺泡巨噬细胞24 h内,糖皮质激素受体表达及活性变化特点.方法将原代培养的大鼠肺泡巨噬细胞分为脂多糖致伤组和地塞米松治疗组,采用RT-PCR和Western Blot在mRNA水平和蛋白质水平测定肺泡巨噬细胞糖皮质激素受体;EMSA测定肺泡巨噬细胞核蛋白中糖皮质激素受体活性.结果脂多糖作用后,糖皮质激素受体mRNA表达下调,24 h恢复正常水平;糖皮质激素受体蛋白质表达降低,4 h降至最低,24 h维持在低水平表达;糖皮质激素受体活性在1 h降到最低,24 h未恢复至正常水平.地塞米松治疗组在治疗后期不能有效维持糖皮质激素受体活性.结论脂多糖(100 ng/ml)作用大鼠肺泡巨噬细胞后,糖皮质激素受体蛋白表达降低,可能与mRNA表达降低及蛋白降解加速有关;糖皮质激素受体活性被显著抑制,出现糖皮质激素抵抗.     

Graves眼病外周血单核细胞糖皮质激素受体表达类型

OBJECTIVE: To study the expression of glucocorticoid receptor (GR) isoform in the peripheral blood mononuclear cell (PBMC) of patients with Graves' ophthalmopathy (GO). METHODS: Semi-quantitative RT-PCR was used to analyze RNA isolated from PBMC of the patients with GO and the normal volunteers. The level of plasma total cortisol (PTC) at 8 a.m. was measured. RESULTS: The hGR alpha/GR beta mRNA ratios of the two groups were 7.58 +/- 5.42 and 14.65 +/- 8.30, respectively. There was significant difference between the two groups (P < 0.05). The PTC levels of the two groups were 304.23 +/- 92.06 and 313.73 +/- 111.05, and no significant difference between them was noted (P > 0.05). No correlation was found between hGR alpha/GR beta mRNA and PTC levels. CONCLUSION: hGR alpha/GR beta mRNA may play a role in the pathogeny of Graves' ophthalmopathy.     

糖皮质激素受体结合域诱饵表达载体的构建及鉴定

目的构建糖皮质激素受体（glucocorticoid receptor,GR）结合域的诱饵表达载体。方法RT—PCR扩增GR结合域,克隆入pMDl8-T,测序正确后,再亚克隆入诱饵载体pGBKT7中,再把构建好的诱饵载体pGBKT7．GR转化到酵母AHl09细胞中,并用Western blot分析诱饵蛋白的表达情况,同时检测诱饵蛋白的毒性和自激活作用。结果成功扩增了GR结合域,并分别成功克隆到pMDl8-T和pGBKT7中,测序结果正确。诱饵载体成功转化到酵母AHl09细胞中,无毒性和自激活作用,Western blot分析结果也证实了酵母细胞高表达诱饵蛋白。结论成功构建了GR结合域的酵母诱饵表达载体。     

Effects of Dexamethasone on glucocorticoid receptor expression in a human ovarian carcinoma cell line 3AO

Ourpreviousworkhasshownthatdexamethasone (Dex) ,asyntheticglucocorticoid ,inhibitshumancarcinomacell(3AO)proliferationandinducesdifferentiationinatime andconcentration dependentmanner 1　Butthemolecularmechanismbehindtheseobservationswasunclear Thepresentstudyinvestigatestheexpressionofglucocorticoidreceptor (GR)in 3AOcellsbyDexaswellastheDex inducedcellulareffectsthatweremediatedbyGR METHODSMaterialsRPMI 16 40mediumwasobtainedfromNissuiPharmaceuticalCo ,Ltd (Tokyo ,Japan) 1,2 ,4 …     

过氧化物增殖激活受体α激动剂非诺贝特下调大鼠肝脏糖皮质激素受体表达

目的 探讨过氧化物增殖激活受体α(PPAR-α)激动剂非诺贝特对大鼠糖皮质激素受体(GR)表达调节作用.方法 30 只雄性SD大鼠随机分为对照组,FE1组(非诺贝特50mg·kg~(-1)·d~(-1),1个月),FE2组(非诺贝特100 mg·kg~(-1)·d~(-1),1个月),FE3组(非诺贝特100mg·kg~(-1)·d~(-1),2个月),MK886组[同时给予非诺贝特(100 mg·kg~(-1)·d~(-1))和PPARa抑制剂MK886(30 mg·kg~(-1)·d~(-1)),1个月].检测SD大鼠肝脏、肌肉、脂肪组织GR基因和蛋白表达水平,同时测定肾上腺11β-羟化酶(CYP11B1)基因表达水平,并以放免法测定大鼠血皮质酮水平.结果 非诺贝特显著下调了大鼠肝脏组织GR基因和蛋门质表达水平,FE1、FE2和FE3 3组GR mRNA水平分别较正常组降低55%、54%、68%,蛋白质表达水平分别降低了28%、77%和99%;并升高.肾上腺CYP11B1表达水平及血中皮质酮水平,而MK886完全逆转了非诺贝特的这些效应[正常组、FE1、FE2、FE3、MK886组皮质酮水平分别为(393±23)、(495±44)、(516±18)、(622±93)、(382±37)ng/ml],提示非诺贝特可抑制肝脏GR表达,并促进肾上腺皮质酮合成增多,这些作用依赖于PPARα激活.结论 PPARα激活剂非诺贝特可抑制肝脏GR表达.     

The glucocorticoid resistance in burn and shock

The changes of glucocorticoid receptor (GR) and the reactivity of target cells toglucocorticoids (GC) were studied experimentally.The results showed that GC resistance,which wascaused mainly by the decrease of GR,developed in burned and shocked rats and dogs.Thesechanges may exacerbate the shock state,and even lead to the development of multiple organfailuue.The protection of GC against intestinal ischemic shock of dogs was demonstrated after theconcentration of dexamethasone (Dex) in plasma was elevated to 10~(-6) mol/L by iv injection of 5mgDex/kg body weight.     

外源性糖皮质激素对胎鼠肺糖皮质激素受体基因的作用

目的：研究糖皮质激素在妊娠后期肺表现活性物质合成过程中对糖皮质激素受体（GR）的调节作用。方法：采用分子生物学杂交技术、Northern印迹法测定胎鼠肺及其二种主要细胞的GR mRNA及Wrang法测定外源性糖皮质激素对胎鼠肺及二种主要细胞GR结合活性的作用。结果：大剂量地塞米松可使胎鼠肺GR结合活性增加GRmRNA无变化。在成纤维细胞（FIB）营养液中加入10^-7mol/L的可的松,GR结合活性和蛋白量增加,GRmRNA无变化。上皮细胞（EPI）GR蛋白量减少,GRmRNA无变化,肺表面活性物质蛋白（SP－A）经同样处理其值增加。结论：胎鼠肺GR的调节发生于转译后水平,妊娠后期使用糖皮质激素对胎鼠的成熟有重要生理意义。     

人参茎叶皂苷对失血性休克大鼠糖皮质激素受体的影响

目的 观察人参茎叶皂昔(ginsenosides，GSS)对失血性休克大鼠糖皮质激素受体(GR)的影响，并分析其作用机制，为研制及时抢救失血性休克患者的天然药物制剂提供实验依据。方法 雄性SD大鼠随机分为失血性休克组和对照组，失血性休克组分别每日ig200，100，50mg／kg GSS水溶液，对照组和模型组ig蒸馏水2mL，共10d。以[^3H]地塞米松为配体，用一点分析法测脑和肝胞液GR结合活性(Rs)、半定量RT—PCR方法测肝胞液GR mRNA水平、放免法测血浆促肾上腺皮质激素(ACTH)和皮质酮(GC)浓度。结果 GSS组大鼠脑和肝胞液的GR结合活性高于单纯失血性休克组，其中以中剂量组最明显(P＜0．01)；GSS组大鼠肝胞液GR mRNA表达水平高于单纯失血性休克组；GSS组大鼠血浆ACTH和GC浓度和单纯失血性休克组没有明显差别。结论 GSS可减轻失血性休克大鼠GR结合活性的下降幅度，作用机制可能与其促进了GR mRNA表达有关，并可能存在一个最佳剂量。     

Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

Background Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting.Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols.Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.     

Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

Background Glucocorticoid (GC) insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor (GR). Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins (hsp) especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting.Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats (30% total body surface area immersion scald) and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols.Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.     

糖皮质激素炎症通路研究现状

糖皮质激素(glucocorticoid,GC)由于其强大的抗炎和免疫抑制等作用被广泛应用于临床.但是,随着对GC认识的深入,研究者们发现GC还能发挥促炎作用,这种效应甚至是机体活动所必须的.机体对GC的反应取决于GC的浓度、机体免疫系统的生理状态、刺激持续时间(急性或慢性)以及疾病的类型等.此外,越来越多的患者出现了...     

BALB/c、C57BL/6小鼠糖皮质激素效应差异机制研究

目的 观察BALB／c和C57BL／6两株小鼠的糖皮质激素(Gc)效应差异，并对其分子机制进行初步探讨。方法 采用放免测定、Western blot和EMSA方法检测两株小鼠即时应激前后血清皮质醇浓度、脑组织糖皮质激素受体(GR)表达和脑组织GRE结合效率。结果 BALB／c和C57BL／6小鼠存在Gc-GR信号通路效应差异，C57BL／6小鼠应激前、后GRE结合效率(P＜0．01)和GR胞核／胞装比值(P＜0．01)均高于BALB／c小鼠，但两株小鼠应激前后血清皮质醇浓度(P＞0．05)和胞装内GR含量(P＞0．05)相差不显著。结论 GR核转位量导致的GRE结合差异在两株小鼠Gc—GR效应差异中扮演了重要角色。     

糖皮质激素受体基因G679S多态性与激素抵抗型哮喘的相关性

目的：检测糖皮质激素受体（Glucocorticoid Receptor,GR）基因G679S的多态性,探讨其与激素抵抗型哮喘的相关性．方法：采用聚合酶链反应-序列特异性引物（PCR-SSP）的方法对217例激素敏感型哮喘患者、64例激素抵抗型哮喘患者及221例健康对照组的GR基因G679S进行多态性分析,判断其与激素抵抗、血清皮质醇浓度及GR解离常数（Kd）的相关性．结果：GG型、GA型及AA型基因频率在激素抵抗组分别为46．9％,42．2％,10．9％．与敏感组的63．6％,31．3％,5．1％和对照组的65．6％,29．0％,5．4％的基因频率有显著差异（P=0．035,P=0．02）．抵抗组GA＋AA基因型及A等位基因频率明显高于其他两组,而抵抗组GA＋AA基因型Kd值显著高于GG型（P=0．029）．血清皮质醇浓度在各基因型无明显差异．结论：G679S基因多态性与哮喘激素抵抗的产生有关,等位基因A可能是激素抵抗产生的易感基因．     

糖皮质激素对兔脑、肺和肝组织糖皮质激素受体的调节

采用放射配基结合分析法，研究地塞米松（Ｄｅｘ）对兔脑、肺、肝的糖皮质激素受体（ＧＲ）的调节作用。结果发现，静脉注射Ｄｅｘ５ｍｇ／ｋｇ后兔肺、肝胞液ＧＲ的结合量明显下降，与对照组比较Ｐ＜０．００１，脑胞液ＧＲ与对照组比较无显著差异。提示糖皮质激素（ＧＣ）对兔不同靶器官ＧＲ的调节存在不同特点。     

基于糖皮质激素受体信号通路中药抗抑郁作用及机制的研究进展

抑郁症发病机制复杂,其典型病理特征是糖皮质激素（GC）水平的异常升高和下丘脑-垂体-肾上腺（HPA）轴功能亢进,主要由GC受体（GR）介导。研究表明,由抑郁症患者GR功能降低和数量减少引起的HPA轴功能亢进是抑郁症的诱因之一。而抗抑郁药可以改善GR的异常变化,提示GR是抗抑郁的一个新靶点。近年来,中药在治疗抑郁症方面取得了很大进展,文章就中药基于GR途径治疗抑郁症的作用及机制进行综述,以期为临床治疗抑郁症提供参考。     

Increased hsp70 of glucocorticoid receptor complex induced by scald and heat stress and its possible effect on the affinity of glucocorticoid receptor

Background Glucocorticoid （GC） insensitivity/GC resistance is an important etiological and prognostic factor in multiple diseases and pathophysiological processes such as scald, shock and asthma. The function of GC was mediated by glucocorticoid receptor （GR）. Scald not only decreased the expression of GR but also reduced the affinity of GR, which played an important role in GC resistance in scalded rats. Whereas the molecular mechanism responsible for the decrease of GR affinity resulted from scald remains unclear. Recent studies showed that the changes of heat shock proteins （hsp） especially hsp90 and hsp70 of GR heterocomplex were associated with GR low affinity in vitro. Methods The affinity of GR in hepatic cytosols and in the cytosols of SMMC-7721 cells were determined by radioligand binding assay and scatchard plot. GR heterocomplex in cytosols were captured by coimmunoprecipation and the levels of hsp90 and hsp70 of GR complex were detected by quantitative Western blotting. Results Similar with that of hepatic cytosol of scalded rats, a remarkable decrease of GR affinity was also found in the cytosol of heat stressed SMMC-7721 cells. The level of hsp70 of GR complex in hepatic cytosol of scalded rats （30% total body surface area immersion scald） and in cytosol of heat stressed human hepatocarcinoma cell line SMMC-7721 were both increased by 1.5 fold, whereas no change of hsp90 in GR heterocomplex was found. According to the correlation analysis, there may be a positive relationship between increased hsp70 of GR complex and decreased GR affinity in the cytosols. Conclusions The primary results indicated that the level of hsp70 of GR heterocomplex was increased in the hepatic cytosol of scalded rats and the cytosol of heat stressed SMMC-7721 cells. The increase of hsp70 of GR complex might be associated with the decrease of GR affinity.