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 …     

地塞米松给药及撤药后糖皮质激素受体的表达

目的 观察地塞米松对体外培养人皮肤角质形成细胞--HaCaT细胞糖皮质激素受体(glucocorticoid recep-tor,GR)α仅和β表达的调节以及撤药后GRα、GRβ基因表达的后续变化,并探讨其临床意义.方法 以含10-6mol/L地塞米松的DMEM(高糖)培养基孵育细胞,48 h后换不含地塞米松的培养摹继续孵育.在地塞米松给药后24、48 h以及撤药后24、48、72 h的不同时相点,采用RT-PCR和Western blot检测GRα、GRβ的表达变化,采用免疫荧光化学法观察GRα、GRβ的细胞内定位.结果 地塞米松给药后24、48 h,GRα的mRNA和蛋白质水平逐渐下降(P<0.05),呈时间依赖性,GRβ的mRNA和蛋白质水平均逐渐上升(P<0.05);撤药后24、48、72 h,GRα和GRβ的上述变化不同程度地向原有水平回复.结论 地塞米松下调GRα并上调GRβ,可部分解释持续外用糖皮质激素制剂后出现的激素耐受现象;下调的GRα和上调的GRβ在地塞米松撤药后呈现向其原有水平恢复的趋势,提示临床上外用激素治疗应避免长期使用.     

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.     

慢性阻塞性肺疾病激素不敏感与糖皮质激素受体核内转移的相关性

 目的  研究慢性阻塞性肺疾病 (chronic obstructive pulmonary disease,COPD)对糖皮质激素不敏感是否与糖皮质激素受体 (glucocorticoids receptor,GRα)的核内转移有关。方法  将来自12例健康人、15例轻中度哮喘患者和15例COPD患者的人外周血单个核细胞 (peripheral blood molecule cells,PBMC)进行体外培养。ELISA方法检测其IL-8蛋白质水平,并计算地塞米松 (Dex)半抑制浓度。PBMC在Dex中培养0.5~6 h,通过Western blot检测GRα蛋白质水平,GRα的核内转移则通过免疫荧光和共聚焦显微镜来观测和分析评估。结果  COPD患者PBMC较轻中度哮喘患者和健康志愿者对Dex相对不敏感。Dex半抑制率 (IC50Dex)与糖皮质激素受体GRα核内转移呈负相关 (r=-0.54,P=0.000 8)。Dex可诱导增加各组PBMC中的总GRα,但GRα胞内分布各组间存在差异。Dex可明显增加健康人及哮喘患者核内的GRα,但并未增加COPD患者核内GRα。健康人及哮喘患者的PBMC中,Dex预处理能显著抑制TNF-α诱导的IL-8分泌,而在COPD患者的PBMC中这种抑制作用不明显。结论  COPD对糖皮质激素的不敏感与GRα核内转移有关。GRα核内转移受抑制可能是造成糖皮质激素在COPD患者中发挥抑制炎性作用欠佳的原因之一。     

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.     

热休克蛋白90和糖皮质激素受体比例失衡对哮喘患者T淋巴细胞凋亡的影响

目的探讨热休克蛋白90(HSP90)与糖皮质激素受体(GR)比例失调对T淋巴细胞凋亡的作用,同时观察地塞米松(Dex)对HSP90和GR mRNA表达的影响.方法选择10例哮喘患者和7例正常对照,利用格尔得霉素(GA)特异性阻断HSP90的作用, 造成功能性HSP90与 GR比例降低,用碘化丙啶(PI)染色流式细胞仪计数凋亡细胞的方法,观察用Dex和GA处理的T细胞凋亡情况;用逆转录聚合酶链反应(RT-PCR)的方法观察Dex和GA处理对哮喘T淋巴细胞HSP 90和GR mRNA 表达的影响.结果 Dex可明显诱导哮喘T淋巴细胞的凋亡(3 3 .8%±3.2% 比 23.2%±1.5%,P<0.01),GA对T淋巴细胞的凋亡无明显影响,却可阻断Dex诱导T淋巴细胞凋亡的作用(24.5%±6.0% 比 33.8%±3.2%, P<0.01);De x对正常人T淋巴细胞的凋亡有诱导作用,但其程度较弱(25.9%±3.5% 比 23.1%±1.5%, P<0.05).Dex可抑制哮喘患者T淋巴细胞HSP90和GR 的表达(分别为1.23±0.16比1.68±0.38 和 0.42±0.06比0.54±0.07, P 均<0.05),GA对 Dex的这一作用有阻断作用,但GA本身对HSP90和GR mRNA的表达无明显影响.结论 HSP90/GR 比例降低可影响Dex诱导的T细胞凋亡;Dex对哮喘患者GR和HSP90mRNA的表达有下调作用,而 Dex的这一作用可被GA所阻断.     

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.     

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。     

Rapamycin Sensitizes Glucocorticoid Resistant Acute Lymphoblastic Leukemia CEM-C1 Cells to Dexamethasone Induced Apoptosis through both mTOR Suppression and Up-Regulation and Activation of Glucocorticoid Receptor*

Objective To explore the role of glucocorticoid (GC) receptor (GR) in rapamycin’s reversion of GC resistance in humanGC-resistant T-acute lymphoblastic leukemia (ALL) CEM-C1 cells. Methods CEM-C1 cells were cultured in vitro and treated with rapamycin at different concentrations with or without 1 μmol/L dexamethasone (Dex). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was performed to assess cell proliferation. The cell cycle and cell apoptosis were analyzed by flow cytometry. The expression of GRα mRNA was determined by real-time quantitative RT-PCR. The expression of GR, p-70S6K, Mcl-1, and Bim proteins was detected by Western blot. Results When incubated with rapamycin at different concentrations, CEM-C1 cells showed significant growth inhibition in a time- and concentration-dependent manner. The growth inhibition was synergistically increased when CEM-C1 cells were treated with rapamycin plus 1 μmol/L Dex. CEM-C1 cells treated with rapamycin alone showed no apparent apoptosis, and were arrested at G0/G1 phase. After the treatment with Dex plus rapamycin, CEM-C1 cells demonstrated apparent apoptosis and increased the cell cycle arrested at G0/G1 phase. Rapamycin combined with Dex up-regulated GRα, phosphorylated GR(p-GR), and pro-apoptotic protein Bim-EL in CEM-C1 cells, but inhibited the expression of p-p70S6K, a downstream target protein ofmTOR (mammalian target of rapamycin). Conclusion After the treatment with rapamycin plus Dex, Dex resistant CEM-C1 cells induce growth inhibition and apoptosis. The underlying mechanism may involve inhibition of the mTOR signaling pathway and also be associated with up-regulation of GR expression and activation of GC-GR signaling pathway.     

糖皮质激素对糖皮质激素受体的调节 Ⅰ.人体肝癌细胞系(SMMC-7721)的研究

建立了糖皮质激素受体(GR)的计数瓶培养测定法。用此法研究了10~(-8)、10~(-7)和10~(-6)mol/L地塞米松对人体肝癌细胞系(SMMC-7721)GR的影响,发现地塞米松使GR减少。去除地塞米松后24h,GR恢复到接近原来的水平。结果证明,糖皮质激素对GR呈现负调节。     

小鼠急性肺损伤后糖皮质激素受体的活性改变与热休克蛋白的关系

目的 研究急性肺损伤(ALI)后机体内糖皮质激素受体(GR)的配体结合活性显著下降与其分子伴侣热休克蛋白(Hsps)改变的关系.方法 以小鼠油酸急性肺损伤为模型,用Western blotting和受体的放射配体结合试验动态观察伤后GR、Hsp90、Hsp70和GR的结合容量和亲和力变化.结果 伤后小鼠肺组织水肿、肺体指数和肺含水量显著升高;GR在伤后1 h升高,然后显著下降,在12 h达最低点;Hsp90和Hsp70在伤后的表达逐渐升高,在12 h达最高点.配体结合试验显示在伤后GR Bmax逐渐下降,Kd值升高,与Hsp90/GR的变化趋势一致.结论 急性肺损伤后的GR配体结合活性的改变可能与Hsp90的变化有关.     

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。     

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。     

人参皂苷Rg1诱导人肝HL-7702细胞糖皮质激素受体报告基因表达的作用

目的探讨人参皂苷Rg1(ginsenoside Rg1,G-Rg1)对人肝HL-7702细胞糖皮质激素受体(GR)的调节作用。方法将含有糖皮质激素受体反应元件(GRE)的荧光素酶报告基因的表达质粒载体pGRE-tk-LUC与内参照基因载体质粒pRL-SV40,通过脂质体介导瞬时转染至人肝HL-7702细胞中,利用双荧光素酶报告基因检测系统,观察报告基因的表达变化;利用Western blotting方法检测药物对细胞GR蛋白表达的影响。结果G-Rg1与地塞米松均可诱导pGRF-tk-LUC报告基因表达,且呈剂量依赖性,而且这种诱导作用可以被糖皮质激素受体阻断剂RU486(Mifepristone)所阻断,G-Rg1对细胞GR蛋白表达有显著的抑制作用。结论G-Rg1在体外能够表现出一些类似糖皮质激素样的生物活性,可能是GR的配体。     

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...     

大鼠肺泡巨噬细胞Bcl-2相关抗凋亡蛋白-1表达变化及其调节糖皮质激素受体功能的研究

目的：在模拟大鼠肺泡巨噬细胞炎症合并糖皮质激素治疗的条件下，阐明Bcl-2相关抗凋亡蛋白-1（BAG-1）的表达变化及其对糖皮质激素受体（GR）功能的调节作用。方法：Western blot检测脂多糖（LPS）和地塞米松（Dex）作用大鼠肺泡巨噬细胞BAG-1表达变化；免疫共沉淀法检测核蛋白中BAG-1与GR相互结合作用；相对荧光素酶法检测GR转录激活活性。结果：LPS和Dex作用后，细胞总蛋白中BAG-1L表达增加，BAG-1S表达无变化；核蛋白中只能检测到BAG-1L，表达量在LPS作用24h内逐渐增加；细胞核内BAG-1L与GR在LPS和Dex作用后存在着相互结合，同时GR转录激活活性逐渐降低，其变化与核蛋白中BAG-1L表达变化呈负相关。结论：LPS和Dex作用大鼠肺泡巨噬细胞BAG-1L表达增加，与GR结合后共同转核，并抑制GR活性。该机制可能是感染条件下糖皮质激素抵抗的重要因素。     

地塞米松对顺铂诱导人肺腺癌细胞株凋亡的抑制作用及其机制

目的 研究地塞米松（Dex）对顺铂（CDDP）引起的人肺腺癌细胞株SPCA/Ⅰ凋亡的抑制作用及其分子机制探讨。方法 体外培养SPCA/Ⅰ细胞,分别加入不同浓度Dex培养24 h后,加入不同浓度CDDP继续培养48 h,采用二甲基溴化四氮唑蓝（MTT）法检测细胞存活率;流式细胞仪检测细胞凋亡;用1 μmol/L Dex刺激SPCA/I细胞,分别于1 h、2 h、4 h、6 h、12 h采用RT-PCR技术,检测SPCA/I细胞中糖皮质激素诱导的蛋白激酶1基因（SGK-1）和丝裂原活化蛋白激酶磷酸酶1（MKP-1）基因的表达;用生物素标记的抗糖皮质激素受体（GR）抗体对SPCA/I细胞中GR行细胞免疫组化检测。结果 SPCA/I细胞经Dex刺激后,对CDDP所致的抑制细胞生长和凋亡呈抵抗作用（P<0.05）。 Dex刺激后的SPCA/I细胞表达SGK-1上调,在12 h时表达达高峰,未检测到MKP-1表达;细胞免疫组化显示SPCA/Ⅰ细胞经Dex刺激后GR表达上调,细胞内GR数目高于对照组（P<0.05）。结论 体外实验结果表明,Dex对CDDP引起的SPCA/I细胞凋亡有抑制作用。其分子机理可能是通过上调细胞内GR数目,增强通路下游SGK1的表达,从而产生抗凋亡作用。