热应激对肝硬化内毒素血症大鼠HSP72表达及致炎细胞因子分泌的影响

目的:探讨热应激诱导肝硬化内毒素血症大鼠热休克蛋白72(HSP72)表达及对致炎细胞因子TNF-α分泌的影响.方法:CCl<,4>诱导的肝硬化SD大鼠及正常饮食的SD大鼠先后给予热应激处理及腹膜腔内注入脂多糖(LPS),另外两组肝硬化SD大鼠及正常饮食SD大鼠仅腹腔内注入LPS作为对照.EUSA检测并比较各组大鼠血浆内毒素、HSP72、TNF-α的含量,RT-PCR检测肝组织HS72 mRNA表达,评价HSP72表达对TNF-α分泌的影响.结果:外源给予LPS能加重肝硬化大鼠内毒素血症.热处理及LPS注入后,肝硬化大鼠及正常大鼠血浆及肝组织HSP72表达均较未热处理组增强,而未热处理的两组大鼠LPS注入后血浆TNF-α含量明显高于热处理组.结论:热应激能促进肝硬化内毒素血症大鼠肝组织及血浆HSP72表达,而抑制TNF-α分泌.     

热应激诱导肝硬化大鼠细胞内外HSP72表达及其对TNF-α分泌的影响

目的 探讨肝硬化大鼠热应激后肝组织细胞内外热休克蛋白72(HSP72)的表达及对致炎细胞因子TNF-α分泌的影响.方法 CCl4诱导的肝硬化SD大鼠及正常饮食的SD大鼠均随机分为热应激组和对照组.ELISA检测并比较各组大鼠血浆内毒素、HSP72、TNF-α的含量,RT-PCR检测肝组织HSP72 mRNA表达,评价HSP72表达对TNF-α含量的影响.结果 各组大鼠腹腔注入LPS后均检测到较高含量的血浆内毒素及TNF-α,尤其肝硬化组升高更为明显(均P<0.01),但热应激组较相应组大鼠血浆TNF-α含量显著降低,而血浆内毒素含量变化不大.热应激后各组大鼠血浆及肝组织HSP72表达均增高,尤其在腹腔注入LPS后升高更为明显(均P<0.01),但肝硬化热应激组却显著低于普通饮食热应激组(均P<0.01).热应激组大鼠血浆TNF-α含量显著低于相应非热应激大鼠组(均P<0.01).结论 热应激处理可能促进肝硬化内毒素血症大鼠肝组织及血浆HSP72表达,而抑制TNF-α分泌.     

热休克蛋白反应对青光眼模型大鼠RGCs中HSP72生成的影响及其作用机制研究

目的 研究热休克蛋白(HSP)反应对青光眼模型大鼠视网膜神经节细胞(RGCs)中热休克蛋白72(HSP72)生成的影响及其作用机制。方法 取64只Wistar大鼠,随机分为正常对照组(正常大鼠)、模型组(青光眼模型)、实验1组(青光眼+腹腔注射硫酸锌)、实验2组(青光眼+热休克反应干预),每周各16只,各组进行相应处理。除正常对照组外,其余三组大鼠右眼均行激光光凝处理,比较大鼠激光处理前后左、右眼的眼压变化;比较各组大鼠实验后不同时间点RGCs平均密度和RGCs中的HSP72抗体水平。结果 各组青光眼大鼠激光处理前后左眼眼压的组内和组间比较,均无显著差异(P 0. 05);模型组、实验1组和实验2组经激光处理后3 d、1周、2周、4周右眼的眼压均明显高于激光处理前(P 0. 05),而组间比较并无显著差异(P 0. 05)。正常对照组实验后不同时间点RGCs中均未见HSP72抗体表达;模型组在激光光凝后2周、4周亦未见HSP72抗体表达;实验1组和实验2组在激光处理后3 d至2周时HSP72抗体水平逐渐升高,到2周时达到高峰,随后有所降低至接近正常水平;实验2组不同时间点HSP72抗体水平较模型组显著升高,较实验1组显著下降(P 0. 05)。相比正常对照组,模型组、实验1组和实验2组经激光处理后3d、1周、2周、4周RGCs平均密度均显著下降(P 0. 05);实验2组不同时间点RGCs平均密度较模型组和实验1组均显著升高(P 0. 05)。结论 在青光眼大鼠模型中,通过HSP反应干预可刺激HSP72在RGCs中的生成,且具有保护大鼠RGCs和视神经功能的作用。     

热休克蛋白72的表达对肾移植缺血再灌注损伤的保护及评价意义

目的通过借助Histidine Tryptophan Ketoglutarate（HTK）保护液在肾移植模型中的器官保护作用，研究肾移植缺血再灌注对热休克蛋白（HSP）72表达的影响。方法18只实验用Sprague Dawley（SD）大鼠随机分为2组，分别进行肾移植。单纯冷藏组（C组）：供体肾脏摘除后，在4℃温度下置于普通Ringer solution中保存10h后进行肾移植；HTK组（H组）：供体肾脏摘除后，在4℃温度下置于HTK solution中保存10h后进行肾移植；肾移植缺血再灌后6h，测定血清尿素氮（BUN）、血清肌酐（Cr）、乳酸脱氢酶（LDH）和天冬氨酸氨基转移酶（AST）；组织样本用于HSP72表达指数（HI）的测定，以了解热休克蛋白表达对肾移植中缺血再灌注损伤的评价作用。结果H组血清BUN、Cr、LDH、AST明显低于C组（P〈0．05）；HSP72的表达H组明显高于C组（P〈0．01）。结论HTK保护液对肾脏有明显的保护作用；缺血再灌注后热休克蛋白的表达标志着细胞的再生，并具有一定程度的内源性保护作用。     

大鼠心肌缺血再灌注损伤与热休克蛋白72的保护作用

目的：观察热休克反应(heat shock response，HSR)后0、24、48、96、192h热休克蛋白72(heat shock protein 72，HSP72)表达水平的变化及再灌注后心肌组织超氧化物歧化酶(superoxide dismutase，SOD)含量、心肌收缩功能变化，探讨HSP72对缺血再灌注心肌保护的可能机制。方法：Wistar大鼠48只．体质量250—300g，雌雄不均，随机分为6组，每组8只。全身高温42℃维持15min制作热休克模型。各组心脏离体逆行灌注．常温下(37℃)缺血25min，再灌注40min。测定缺血前、再灌注后心肌组织SOD含量，心肌收缩功能，观察各组心肌HSP72表达。结果：实验组再灌注后24h和48h心肌组织SOD活性分别为(22．37&;#177;4．75)、(23．39&;#177;4．86)Nu／mg，明显高于对照组。对照组和热预处理后0 h几乎无HSP72表达(面密度)，其表达高峰在热预处理后24，48h。分别为137．89&;#177;25．25，146．09&;#177;28．34，随后逐渐降低，于192h返回基线水平。HSP72表达与再灌注后心肌组织SOD(r=0．9234)，心功能恢复率LVSP(r=0．9012)呈显著正相关(P&;lt;0．05)．结论：热休克蛋白能提高抗氧化酶活性，减轻心肌缺血再灌注损伤。     

大鼠心肌缺血再灌注损伤与热休克蛋白72的保护作用

目的:观察热休克反应(heatshockresponse,HSR)后0、24、48、96、192h热休克蛋白72(heatshockprotein72,HSP72)表达水平的变化及再灌注后心肌组织超氧化物歧化酶(superoxidedismutase,SOD)含量、心肌收缩功能变化,探讨HSP72对缺血再灌注心肌保护的可能机制。方法:Wistar大鼠48只,体质量250～300g,雌雄不均,随机分为6组,每组8只。全身高温42℃维持15min制作热休克模型。各组心脏离体逆行灌注,常温下(37℃)缺血25min,再灌注40min。测定缺血前、再灌注后心肌组织SOD含量,心肌收缩功能,观察各组心肌HSP72表达。结果:实验组再灌注后24h和48h心肌组织SOD活性分别为(22.37±4.75)、(23.39±4.86)Nu/mg,明显高于对照组。对照组和热预处理后0h几乎无HSP72表达(面密度),其表达高峰在热预处理后24,48h,分别为137.89±25.25,146.09±28.34,随后逐渐降低,于192h返回基线水平。HSP72表达与再灌注后心肌组织SOD(r=0.9234),心功能恢复率LVSP(r=0.9012)呈显著正相关(P<0.05).结论:热休克蛋白能提高抗氧化酶活性,减轻心肌缺血再灌注损伤。     

大鼠热休克蛋白72基因真核表达载体构建及在COS7细胞中的表达

背景：体外克隆、表达热休克蛋白,尤其正常时少量或不表达,而应激时大量表达的热休克蛋白72对研究其缺血再灌注损伤中的作用尤为重要。目的：构建热休克蛋白72基因真核表达载体并于COS7细胞内表达,为HSP72蛋白免疫调节功能的研究奠定基础。方法：采用RT-PCR技术从BABL/C大鼠肝细胞中扩增出热休克蛋白72cDNA,经限制性核酸内切酶消化后,插入真核表达载体pcDNA3.1（＋）的相应酶切位点,并将重组质粒转染COS7细胞,进行基因表达鉴定。结果与结论：重组质粒插入基因序列检测证实为热休克蛋白72cDNA,并能在COS7细胞稳定表达。成功构建热休克蛋白72真核表达载体,并于COS7细胞中成功转录与表达。     

大鼠热休克蛋白72基因真核表达载体构建及在COS7细胞中的表达

背景:体外克隆、表达热休克蛋白,尤其正常时少量或不表达,而应激时大量表达的热休克蛋白72对研究其缺血再灌注损伤中的作用尤为重要.目的:构建热休克蛋白72基因真核表达载体并于COS7细胞内表达,为HSP72蛋白免疫调节功能的研究奠定基础.方法:采用RT-PCR技术从BABL/C大鼠肝细胞中扩增出热休克蛋白72 cDNA,经限制性核酸内切酶消化后,插入真核表达载体pcDNA3.1(+)的相应酶切位点,并将重组质粒转染COS7细胞,进行基因表达鉴定.结果与结论:重组质粒插入基因序列检测证实为热休克蛋白72 cDNA,并能在COS7细胞稳定表达.成功构建热休克蛋白72真核表达载体,并于COS7细胞中成功转录与表达.     

谷氨酰胺对呼吸机所致肺损伤中HSP70表达的影响

目的 探讨谷氨酰胺(Gln)对呼吸机所致肺损伤(VILI)肺组织HSP70表达的影响及与细胞因子TNF-α的关系.方法 建立正常条件通气及致伤通气大鼠肺损伤模型.雄性SD大鼠40只,随机分五组:对照组、致伤通气组、致伤通气预先给药(Gln)组(C1),致伤通气即时给药组(C2),致伤通气2 h给药组(C3),分别于通气开始前、通气开始即刻及通气后2 h静脉注射谷氨酰胺0.75 g/kg,所有动物通气4 h后经颈动脉放血处死,采用Western bolt的方法检测各组大鼠HSP70的表达.结果 大鼠在致伤通气早期(通气前预先和通气开始时即刻)静脉给予谷氨酰胺可使肺组织HSP70的表达明显增加.结论 对呼吸机所致肺损伤的大鼠静脉注射谷氨酰胺0.75 g/kg可诱导肺组织HSP70的产生,保护肺组织减轻损伤,早期用药效果更优.     

抗热休克蛋白72多克隆抗体血清的制备和检测

目的 探讨在大肠杆菌中表达人热休克蛋白72（hHSP72）与组氨酸（His）的融合蛋白并制备其抗体血清的方法。方法 将hHSP72片断插入His融合表达载体pPROEX-1^TM，重组载体酶切鉴定后，在大肠杆菌中经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达获得His-hHSP72融合蛋白。采用纯化后的HSP72免疫新西兰白兔，制备抗血清；蛋白质免疫印迹法（Western blot）检测重组抗原的免疫活性。结果 重组质粒酶切鉴定结果表明，hHSP72基因已正确插入到pPROEX-1^TM中，经IPTG诱导后，表达出分子质量约为73ku的蛋白，获得了效价为1：100000的多克隆抗体。Western blot检测证明，所制备的多克隆抗体可以与hHSP72特异性结合，其效价高于市售的单克隆抗体。结论 用hHSP72片断在大肠杆菌中能成功表达，并能制备得到多克隆抗体；这种多克隆抗体是一种新的高特异性和高灵敏度的试剂。     

Correlation between clinicopathology and expression of heat shock protein 72 and glycoprotein 96 in human gastric adenocarcinoma

Heat shock protein 72 (HSP72) and glycoprotein 96 (gp96) are highly expressed in cancer tissues. Recent studies indicate the possible roles of HSP72 and gp96 in the development and progression of gastric carcinomas but detailed information is still ambiguous. In this study, we investigated the correlation between clinicopathology and expression of HSP72 and gp96 in human gastric carcinoma. The expression of HSP72 and gp96 was studied in 60 human gastric carcinomas with or without metastasis as well as in mucous membrane adjacent to cancers by way of immunohistochemistry. HSP72 immunoreactivities were detected in 54 of 60 primary tumors (90.0%) and in 22 of 60 mucous membranes adjacent to cancers (36.7%). Likewise, gp96 immunoreactivities were detected in 49 cases of gastric carcinoma (81.7%) and in 15 samples of mucous membrane adjacent to cancer (25.0%). Both HSP72 and gp96 were stained in cytoplasm. HSP72 and gp96 expression in colonic carcinomas with metastasis was significantly higher than those with non-metastasis (p < 0.05). The results indicate that there exists a significant correlation between the expression of HSP72 and gp96 and the progression of gastric carcinomas. The high-level expression of HSP72 and gp96 may be used as diagnostic or prognostic markers for gastric carcinoma.     

Increased inducible heat shock protein 72 expression associated with PBMC isolated from patients with haematological tumours

Abstract

Background. Heat shock protein 72 (Hsp72) is a highly inducible stress protein and molecular chaperone. Cancers have been shown to be associated with increased Hsp72 expression within the tumour itself and this may lead to resistance to apoptosis. Methods. Peripheral blood mononuclear cells (PBMC) were isolated from patients diagnosed with chronic lymphocytic leukaemia (CLL) (n =?27) and chronic myelomonocytic leukaemia (CMML) (n =?16) and Hsp72 expression was characterized on both the cell surface and intracellularly by flow cytometry. To allow for comparison PBMC from breast cancer patients (n =?25) and healthy volunteers (n =?19) were included. Results. Both lymphocytes and monocytes from CLL and CMML patients showed high levels of total Hsp72 expression (4–6 fold increase) in comparison to breast cancer and healthy subjects. The majority of Hsp72 in these tumours was determined to be cell-surface expressed (64–93% of cell total Hsp72). Conclusions. A correlation was observed between lymphocyte and monocyte total Hsp72 expression (p <?0.001) suggesting a common stress response pathway may exist in these blood cells and there are stress conditions present within the circulation. Hsp72 expression was not found to be related to white blood cell count.     

Increased inducible heat shock protein 72 expression associated with PBMC isolated from patients with haematological tumours

Abstract Background. Heat shock protein 72 (Hsp72) is a highly inducible stress protein and molecular chaperone. Cancers have been shown to be associated with increased Hsp72 expression within the tumour itself and this may lead to resistance to apoptosis. Methods. Peripheral blood mononuclear cells (PBMC) were isolated from patients diagnosed with chronic lymphocytic leukaemia (CLL) (n =?27) and chronic myelomonocytic leukaemia (CMML) (n =?16) and Hsp72 expression was characterized on both the cell surface and intracellularly by flow cytometry. To allow for comparison PBMC from breast cancer patients (n =?25) and healthy volunteers (n =?19) were included. Results. Both lymphocytes and monocytes from CLL and CMML patients showed high levels of total Hsp72 expression (4-6 fold increase) in comparison to breast cancer and healthy subjects. The majority of Hsp72 in these tumours was determined to be cell-surface expressed (64-93% of cell total Hsp72). Conclusions. A correlation was observed between lymphocyte and monocyte total Hsp72 expression (p 相似文献   

Interaction between heat shock protein 72 and alpha-fetoprotein in human hepatocellular carcinomas

BACKGROUND: AFP in adult serum often signals pathological conditions, particularly the presence of hepatocellular carcinoma (HCC) and germ cell tumors containing yolk sac cell elements. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to overexpress in epithelial carcinoma cells. There may be a possible correlation between the expression of HSP72 and AFP during the growth and differentiation of hepatocellular carcinoma cells. We investigated the interaction between heat shock protein 72 (HSP72) and alpha-fetoprotein (AFP) in human hepatocellular carcinomas. METHODS: The expression and localization of HSP72 and AFP in human hepatocellular carcinomas were determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and AFP in hepatocellular carcinoma cells was analyzed by immunoprecipitation and Western immunoblots. RESULTS: Hepatocellular carcinoma synchronously co-expressed higher level of HSP72 and AFP than in adjacent normal liver tissues. HSP72 were stained in cell nuclei and cytoplasm respectively, while AFP stained in cell plasma. Based on Western blotting methods, AFP was detected in the immunoprecipitate of anti-HSP72 monoclonal antibody (mAb), while HSP72 existed in the immunoprecipitate of anti-AFP mAb. CONCLUSIONS: HSP72 and AFP expression are higher in hepatocellular carcinoma tissues. HSP72 was associated with alpha-fetoprotein in human hepatocellular carcinoma cells. The interaction between HSP72 and AFP in human hepatocellular carcinoma cells can be a new route for studying the pathogenesis and immunotherapy of hepatocellular carcinoma.     

Leptin and stress protein (heat shock protein 72: HSP72) in patients with obstructive sleep apnea-hypopnea syndrome

Leptin is a circulating hormone that is expressed abundantly and specifically in adipose tissue. Leptin induces a complex response involving control of body weight, energy expenditure and fat distribution. It is difficult for patients with obstructive sleep apnea-hypopnea syndrome to reduce and maintain their weights. Therefore, it is important to understand the circadian rhythms and regulation of serum leptin levels in order to control the body weight of obstructive sleep apnea-hypopnea syndrome (OSAHS) patients. Heat shock protein(HSP) 72 is generally known to be a stress-inducible isoform that is barely detectable under unstressed conditions but which is rapidly synthesized during or after stress. Recent data suggest that OSAHS may have significant effects on the serum leptin levels and HSP72 levels in peripheral blood mononuclear cells(PBMC) of patients with OSAHS.     

Continuous and pulsed ultrasound do not increase heat shock protein 72 content.

Therapeutic ultrasound (US) is a common treatment used in the rehabilitation of injured muscle. To determine whether therapeutic US could increase the content of heat shock protein (HSP) 72 in skeletal muscle, male Sprague-Dawley rats were anesthetized and the muscles from one hind limb treated with 15 min of US at 1 MHz using either: 1. continuous US at 1.0 W/cm(2), 2. pulsed US at 2.0 W/cm(2) at 50% duty cycle, or 3. pulsed US at 1.0 W/cm(2) at 20% duty cycle. All treatments were applied using a transducer (1.6-cm diameter) on an area of the rat hind limb twice the size of the sound head. At 24 h following treatment, the plantaris, soleus, white and red gastrocnemius muscles were removed and assessed for HSP 72 content by Western blotting. No significant increases in HSP 72 content were detected in any of the muscles examined following any US treatment. These results suggest muscle HSP content is not elevated following a typical therapeutic dose of either continuous or pulsed US in the rat.     

超声微泡造影剂介导HSP72 siRNA基因转染对大鼠缺血-再灌注肝损伤的作用

笔者前期的研究中发现,缺血-再灌注肝损伤中过度表达的热休克蛋白72 (heat shock protein 72,HSP72)可以释放至细胞外,并作为Toll样受体的内源性配体启动炎性应答而导致肝组织细胞损伤[1-2];HSP72 siRNA能抑制体外热应激肝细胞释放HSP72[3].本研究利用超声微泡造影剂结合HSP72siRNA,并靶向转染入肝组织,观察HSP72siRNA在肝组织细胞内的表达及对肝组织细胞损伤的影响.