光亲和标记测定烫伤大鼠肝胞液糖皮质激素受体分子量

本实验室以往的工作证明,大鼠烫伤后,不但有肝胞液糖皮质激素受体结合容量的改变,而且有表观解离常数的增大和糖皮质激素受体沉降系数的改变。本文采用[~3H]去炎松缩酮光亲和标记法测定了烫伤大鼠肝胞液糖皮质激素受体的分子量,和正常大鼠无显著差异,因而说明,受体沉降系数的改变不是由于受体结合亚单位分子量的改变所致。     

大鼠严重烫伤早期应激反应中肝脏糖皮质激素受体变化的实验研究

目的 研究大鼠在严重烫伤早期应激反应中肝脏糖皮质激素受体（GR）的表达变化及意义，探讨影响GR改变的分子机制。方法 通过免疫印迹分析GR在严重烫伤后早期不同时相点肝脏中的表达变化及烫伤血清、细胞因子以及高浓度地塞米松对体外培养肝细胞GR表达的影响；应用放免和ELISA方法测定烫伤大鼠血清皮质酮和细胞因子TNF－α、IL-β的浓度变化。结果 大鼠肝脏GR的表达在严重烫伤后早期显著下调，而血清皮质酮和血清炎性细胞因子在烫伤早期均显著升高，体外实验则证实烫伤血清和TNF－α、IL－1对培养肝细胞的GR的表达有明显抑制作用，高浓度地塞米松作用则不明显。结论 严重烫伤会引起大鼠肝脏GR表达显著下调，烫伤早期分泌增多的炎性细胞因子可能是引起GR下调的主要原因之一，而烫伤早期机体内部大量生成的糖皮质激素对GR的表达并无显著影响。     

糖皮质激素受体在创伤失血性休克大鼠肝损伤中的作用

目的 从组织受体水平探讨糖皮质激素受体(GR)在创伤失血性休克后肝组织中的变化及其对肝损伤的作用机制.方法 采用双侧股骨骨折伴失血性休克模型,并对GR进行阻断后再致伤,动态观察伤后8h大鼠肝组织GR、肝脏病理、肝功能生化指标、伤后8h大鼠死亡率等变化.肝组织GR采用免疫印迹法测定其蛋白含量,并进行计算机图像分析.结果 肝组织GR的蛋白含量在创伤失血性休克后1h即开始下降,2h明显低于正常对照组(P＜0.01),6h降至最低,8h仍显著低于正常;光镜下伤后4h-8h肝窦内少许淤血,有散在炎性细胞浸润;血清丙氨酸氨基转移酶(ALT)、总胆红素(TB)伤后4h开始增高,白蛋白下降;大鼠死亡率10%.GR阻断后再致双侧股骨骨折并失血性休克,光镜下伤后2h肝窦内即可见较多炎性细胞浸润,血清ALT、TB在伤后2h即有明显升高(P＜0.01),白蛋白明显下降(P＜0.01);大鼠死亡率明显升高(50%).结论 GR不足可导致严重创伤并失血性休克后肝损伤的发生;且减少越多,肝损害越重,死亡率越高.提示GR在严重创伤休克后肝组织细胞损伤与抗损伤机制方面可能起着重要作用.     

丹参对大鼠烫伤早期肝组织含水量的影响

目的观察大鼠严重烫伤早期肝组织含水量的变化及丹参对它们的影响。方法建立30%III度烫伤模型,88只Wistar大鼠随机分为正常对照组(A组,8只)、平衡液复苏组(B组,40只)和丹参治疗组(C组,40只)。B、C两组烫伤后立即按Parkland公式腹腔注射平衡液120ml/kg,C组在同样治疗的基础上腹腔注射丹参注射液3ml/kg,B、C两组分别于伤后3,6,12,24,48h测定肝组织含水量。结果与对照组相比较,复苏组肝组织含水量在伤后6h即明显高于对照组,48h达高峰;丹参治疗组在各相应时相点与对应的复苏组比较,两者的变化趋势基本一致,但肝组织含水量均低于复苏组,伤后6h起显著低于复苏组。结论(1)大鼠严重烫伤早期肝组织含水量即明显升高。(2)丹参可使严重烫伤早期肝损伤的肝组织含水量降低,对烫伤早期肝损伤有保护作用。     

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。     

肝素保护肠黏膜屏障的实验研究

目的 探讨肝素对烫伤鼠肠黏膜屏障的影响.方法 雄性Wistar大鼠70只(体重200~250 g)随机分为无烫伤组10只,烫伤组及烫伤后使用肝素组(肝烫组)各30只.用沸水制成大鼠背部35%Ⅱ°烫伤,肝烫组于烫伤后立即、4 h、8 h皮下注射硫酸肝素100μ·kg-1,烫伤组和无烫伤组相同时相皮下注射等量生理盐水.烫伤组、肝烫组分别于烫伤后8 h、12 h、24 h处死动物,无烫伤组8 h全部处死.大鼠FITC-D灌胃测定肠粘膜通透性,穿刺腹主动脉测血浆IL-6、IL-8.结果 烫伤后大鼠门静脉FITC-D标记的葡聚糖及血浆IL-6、IL-8与正常组相比均显著升高(P<0.01).使用肝素后,门静脉FITC-D标记的葡聚糖及血浆IL-6、IL-8均明显下降,分别与烫伤组8 h、12 h、24 h相比有显著差别.结论 烫伤可导致大鼠肠粘膜损伤,肠粘膜通透性增加,血浆IL-6、IL-8升高;肝素可以保护应激状态下的肠粘膜屏障,抑制炎症因子IL-6、IL-8 的产生,减轻全身炎症反应,保护脏器功能.     

生脉散组分配伍对热损伤大鼠糖皮质激素受体的影响

目的：研究生脉散组分配伍对热损伤大鼠糖皮质激素受体（glucocorticoid receptor,GR）的影响。 方法：雄性SD大鼠32只随机分为正常对照组、模型组、人参总皂苷组和生脉散组分配伍组（简称组分配伍组）,每组8只,连续灌胃1周进行预处理。除正常对照组外,其余各组大鼠于末次给药30min后复制大昂热损伤模型。随后立即断头处死动物,收集血清和肝、肺、肾脏组织。酶联免疫吸附测定法（enzyme-linked immkinosorbent assay,ELISA）检测血清皮质酮（corticosterone,CS）的浓度,放射免疫检测法（radioimmunoassay,RIA）检测血清促肾上腺皮质激素（adrenocorticotropic hormone,ACTH）的浓度;放射性配体受体结合法（radioligand receptor binding assay,RRA）检测肝、肺、肾细胞液GR结合容量。 结果：模型组肝、肺、肾胞液GR明显低于正常对照组（P〈0.01）。组分配伍组肝和肺胞液GR结合容量明显高于模型组（P〈0．01）,肾胞液GR结合容量与模型组比较,差异无统计学意义。组分配伍组肝胞液GR明显高于人参总皂苷组（P〈0．01）,肺和肾胞液GR结合容量与人参总皂苷组比较有上升的趋势．但差异无统计学意义。正常大鼠CS及ACTH水平与模型组、组分配伍组和人参总皂苷组比较,差异有统计学意义（P〈0．01）,模型组CS及ACTH水平与各给药组比较,差异无统计学意义。 结论：生脉散组分配伍可增强人参总皂苷上调热损伤大鼠GR水平的作用。     

未去肾上腺大鼠肝胞液糖皮质激素受体的测定及烫伤后的变化

在内源性激素存在的条件下,用交换法测定了大鼠肝胞液的糖皮质激素受体。交换条件为Oc4.5～5小时。在这条件下,[~3H]皮质酮—受体复合物的解离将近90％。向去肾上腺大鼠肝胞内预先加入不同量的皮质酮保温后,再测定[~3H]地塞米松特异结合,其结果表明,胞液皮质酮浓度在6nm以内时,[~3H]地塞米松特异结合容量(Ro)的测定值的减少不超过10％,表面上的解离常数(Kd)的测定值基本上不变。用这方法测定烫伤大鼠肝胞液的Ro和Kd,结果Ro减少。烫伤后1、12、24、36小时和5天的Ro分别为对照组的67.4±15.2、70.6±22.0、79.7±5.8、113.0±24.8和102.9±17.6％(均值±标准差)。烫后1小时Kd增大。对其可能的机理作了讨论。     

严重烫伤大鼠肝脏热休克蛋白90α的表达变化及意义

目的 研究严重烫伤早期大鼠肝脏组织中糖皮质激素受体分子伴侣热休克蛋白90α(HSP90α)的表达变化及意义。方法 用免疫印迹技术分析严重烫伤后不同时相肝脏组织中HSP90α的表达量改变，通过免疫组化进一步观察HSP90α在肝细胞胞核中的表达变化。结果 大鼠严重烫伤后72h内肝脏HSP90α的表达明显升高，尤其以12～48h更为显著，在48h左右肝细胞核内还出现HSP90α的异位高表达。结论 严重烫伤可以引起肝脏HSP90α异常高表达，这可能是严重创伤早期糖皮质激素受体表达和功能发生改变的原因之一。     

甲状腺激素对大鼠肝糖原及肝胞液糖皮质激素受体的影响

用实验性甲状腺功能亢进大鼠,同时观察甲状腺激素对肝糖原含量的影响和肝胞液糖皮质激素受体(GCR)的变化。结果表明,甲状腺片(75 mg/kg·d,ig×10 d和100 mg/kg·d,ig×10d)所致甲状腺功能亢进,可同时引起与甲状腺片剂量有依赖关系的肝糖原减低和与之平行的肝胞液GCR数目升高。提示,甲状腺激素减低大鼠肝糖原含量的作用与肝胞液GCR的增高有直接关系。     

Determination of the Molecular Weight of Photoaffinity Labeled Glucocorticoid Receptor in Scalded Rat Liver Cytosol

Previous work in our laboratory showed that,after scalding,there wasnot only a decrease of rat liver cytosol [~3H]-Dexamethasone specific binding sitesbut also an increase of apparent dissociation constant of [~3H]-Dex and change ofsedimentation coefficient of glucocorticoid receptor.The present study was carriedout using photoaffinity labeling of glucocorticoid receptor in rat liver cytosol.Itwas found that there was no significant difference between molecular weights ofglucocorticoid receptor of normal and scalded rats determined by SDS-polyacrylamide gel electrophoresis.Our result suggests that chnage ofsedimentation coefficient is not due to alteration of molecular weight ofglucocorticoid receptor binding subunit.     

驻极体对烫伤大鼠肺血管壁通透性变化的影响

通过检测肺组织匀浆中荧光标记白蛋白的含量，观察了大鼠烫伤６，１２，２４ｈ后肺血管壁通透性的变化及－１００、－２５０，－５００，－７５０，－１０００Ｖ驻极体对这种变化的影响。     

NMDA受体对烫伤应激大鼠海马糖皮质激素受体基因表达的影响

目的：观察烫伤应激后海马糖皮质激素受体（GR）基因表达的变化，探讨N－甲基－D－天冬氨酸（N－methyl-D-aspartate,NMDA）受体在这种变化中的作用。方法：利用RT－PCR技术，检测烫伤前腹腔注射NMDA受体拮抗剂MK－801及激动剂NMDA对烫伤应激后2h GR mRNA水平的影响。结果：烫伤应激后2h海马GR mRNA水平明显降低（P＜0．01）；应激前腹腔注射MK－801使烫伤后降低的GR mRNA水平明显恢复（P＜0．01），注射NMDA使其进一步降低（P＜0．05），注射生理盐水无明显变化。结论：NMDA受体介导了烫伤应激导致的海马GR转录水平基因表达的下降。     

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.     

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.     

热应激对大鼠肝组织及人前列腺癌PC-3细胞RhoB表达的影响

目的:观察烫伤后大鼠肝脏组织及热应激后前列腺癌PC-3细胞小G蛋白RhoB表达的变化,探讨热应激对体内外RhoB表达的影响.方法:制备雄性SD大鼠30%体表面积Ⅲ度背部烫伤模型,烫伤后2、4、8、16 h取相应大鼠肝脏组织(n=6),应用RT-PCR和Western印迹分析测定RhoB mRNA和蛋白水平,并以正常大鼠肝脏组织作对照(n=6).制备人前列腺癌PC-3细胞热应激模型,热应激后0.5、1、2、4、8 h取细胞检测RhoB的表达,并以未处理细胞作对照.结果:烫伤后4 h,大鼠肝组织RhoB mRNA表达量最高,为正常对照的3.2倍(P＜0.01),8 h开始下降;而RhoB蛋白表达量在烫伤后8 h最高(P＜0.01) .PC-3细胞RhoB mRNA在热应激后2 h时开始上调;4 h时达到最大值,约为对照的2.8倍(P＜0.01);热应激后8 h RhoB蛋白表达量最高(P＜0.01).结论:在体内、体外实验中热应激均能上调小G蛋白RhoB mRNA和蛋白的表达.     

Relationship between the state of reduction-oxidation and protein degradation in the soleus muscle after scalding in rats

One hind leg(about 7% TBSA)of a rat was scalded and the changes of thereduction-oxidation state and protein degradation in the soleus muscle were observed inthe 72nd h after scalding both in vitro and in vivo.It was found that the lactate/pyruvate(L/P)and malate/pyruvate(M/P)ratios in the soleus muscle were significantly lower andthe protein degradation rate significantly higher in the scalded rats than those in the controland in the unscalded legs.After the addition of insulin to the medium,significant eleva-tion of L/P and M/P ratios and reduction of the protein degradation rate were observedin the soleus muscle.These findings suggest that there is a good correlation between thechanges of the reduction-oxidation and the protein degradation rate in the cytosol of thesoleus muscle after scalding in rats.     

The role of interleukin--6 in protein degradation of skeletal muscles early after severe scalding in mice

ItwaswellacceptedthattheintensificationofproteindegradationaftertraumatamainlyresultsfromtheelevationofglucagonandglucocorticoidsL'j.Recentlyitwasreportedthatinflammationmediatorssuchasvariouscytokinesalsoexerttheireffectsonthepostraumatichypermetabolicstage.Tumornecrosisfactorandinterleukin--lhavebeenconfirmedtotakepartinproteinmetabolisminthetissuesdamagedintrauma[2'3].Interleukin--6(IL--6)levelwasfoundelevatedaftertrauma[4],whichwasinducedbyTNFandIL--115j.Thisstudywasdesignedtoobserveth…