磷酸化p38在高温处理后金黄地鼠胚心脏的表达

目的研究心脏正常发育和异常分化过程中磷酸化p38(p-p38)的表达变化,探讨p38在高温处理后金黄地鼠胚胎心脏发育中的作用。方法将金黄地鼠孕鼠于受孕8d置42℃水浴持续20min,于高温处理后12h、24h、36h、48h剖腹取胎,制备石蜡切片。利用免疫荧光染色技术,观察心脏正常发育和异常分化过程中磷酸化p38(p-p38)的表达变化。结果 p-p38的免疫阳性产物表达于鼠胚内皮细胞及心胶质细胞胞浆中,高温处理后不同时间点实验组p-p38的表达均较对照组明显降低。结论高温可导致金黄地鼠胚胎心脏p-P38表达减弱,p-p38对心脏的正常发育具有重要作用。     

榄香烯或热休克对人肝癌细胞HepG2HSP70膜表达及多种HSP基因表达的影响

目的 :研究榄香烯或热休克对人肝癌细胞HepG2 HSP70膜表达及多种HSP基因表达的影响。方法 :免疫荧光和FCM观察榄香烯 (5 0 μg ml,1h)或热休克 (42℃ ,1h)处理后肿瘤细胞HSP70的膜表达 ,放线菌素D阻断基因转录。应用人类基因表达谱芯片分析经榄香烯或热休克处理的人肝癌细胞HepG2 多种热休克蛋白基因及与热休克蛋白调控相关基因表达谱的改变。结果 :榄香烯或热休克处理 1h后 ,肿瘤细胞膜表面HSP70的表达均有增高 ,而以榄香烯处理为明显 ,放线菌素D的加入在两种处理中均增高了HSP70膜表达的阳性率。两种处理均使细胞的HSP70HP(EnhancerProtein)基因表达增高 ,而以热休克处理为更明显 ,HSPA2的表达均有所下降 ,也以热休克处理更明显 ,HSF1基因在热休克处理为上调 ,而在榄香烯处理则为下调 ,与肿瘤免疫密切相关的HSP70、HSP72、HSP75及HSP90、gp96基因的表达则没有变化。结论 :榄香烯较热休克处理早期能更多地促进HepG2 细胞HSP70的膜表达 ,其机制可能是与其改变胞内已存在的HSP70的分布 ,和 或促进HSP70mRNA的翻译有关。两种处理均能改变与HSPs调控相关基因的表达 ,并可引起HSPA2基因表达下调。     

麻醉对小鼠热休克反应的影响

目的 通过分析麻醉状态和清醒状态小鼠在相同热休克条件下的热休克反应异同，了解整体调控在生物热休克反应中的意义和作用。方法 经40、42、44和46℃，分别处理清醒状态和麻醉状态小鼠30min，于正常饲养条件下恢复24h后，检测其肝脏热休克蛋白70(HSP70)的表达差异及酸性和中性蛋白水解酶活性变化。结果 当热休克温度为44～46℃时，清醒状态小鼠肝脏的HSP70合成能力下降，而麻醉小鼠肝脏保持较强的HSP70合成能力；两组小鼠肝脏的酸性蛋白水解酶活性基本与HSP70合成正相关，中性蛋白水解酶活性基本与HSP70合成负相关。结论 麻醉状态小鼠肝脏的热休克耐受性大于清醒小鼠；小鼠肝脏的热休克反应受整体和细胞两个水平调控，并涉及除HSP70合成以外的其他生化活动。     

热休克蛋白70对离体心脏心肌间质的保护作用

目的探讨热休克蛋白70（HSP70）对大鼠离体心脏心肌间质的影响。方法Wistar大鼠16只，分为2组：对照组（C，n=8），腹腔注射生理盐水0．4ml，24h后取离体心脏灌注HTK心脏保护液，4℃保存3h后建立Langendorff灌注模型，灌注KH液2h；实验组（E，n=8），腹腔注射重酒石酸去甲肾上腺素，24h后取离体心脏，方法同对照组。以心肌细胞中HSP70含量、血流动力学指标、心肌组织羟脯氨酸（HP）、内皮索（ET）含量和心肌超微结构等作为观察指标。结果HSP70含量E组与C组比较明显增高；E组心功能恢复方面优于C组（P〈0．05），HP含量优于C组（P〈0．01），ET含量低于C组（P〈0．01），心肌超微结构损伤较C组明显减轻。结论HSP 70对供心心肌间质具有保护作用。     

小鼠不同组织器官热休克蛋白70表达的研究

目的　研究热休克恢复期小鼠大脑、垂体、肾、肾上腺中HSP70的表达规律。　方法　昆明系小鼠随机分为对照组和实验组。实验组动物 :1 分别以 40℃ ,42℃ ,44℃ ,46℃热休克处理 30min ,恢复 2h和 8h ;2 以46℃热休克处理 30min ,恢复 2、4、6、8、12、2 4、48、72h ,以免疫组织化学结合图像分析技术观察HSP70的表达。　结果　 1 42℃ ,44℃ ,46℃均能诱导肾、肾上腺表达HSP70 ,44℃、46℃可诱导大脑、垂体表达HSP70 ,但均以 46℃组阳性免疫反应面积最大 (P <0 0 1) ;2 HSP70的合成高峰在大脑和垂体为热休克恢复期 4～ 8h(P <0 0 1) ,肾和肾上腺为 8～ 12h(P <0 0 1) ;3 HSP70阳性免疫反应定位于大脑神经膜和郎飞结 ,垂体神经膜 ,肾小管、肾上腺网状带和束状带细胞质 ,肾中HSP70阳性免疫反应肾小管较肾小球强 ,肾髓质较肾皮质强。　结论　热休克反应中不同组织器官HSP70的表达存在明显差异性 ;HSP70合成迅速且降解缓慢 ;大脑和垂体有较强的耐热能力     

急性心肌梗死猝死者心肌细胞hsf1和HSP70改变的意义

目的探讨急性心肌梗死猝死者梗死区心肌细胞内热休克转录因子1（hsfl）和热休克蛋白70（HSP70）改变的临床意义。方法分别用RT-PCR和免疫组化法检测（IHC）18例急性心肌梗死猝死者（研究组）和15例心脏正常因车祸快速死亡者（对照组）心肌细胞中hsfl和HSP70基因的mRNA和蛋白表达量。结果急性心肌梗死猝死者心肌细胞hsfl和HSP70的mRNA表达量都显著高于正常对照组（P〈0.01），且hsfl和HSP70mRNA的表达量之间呈显著的正相关关系（P〈0．001）。急性心肌梗死猝死者心肌细胞hsfl和HSP70蛋白在细胞浆和细胞核表达较对照组显著增强（P〈0.001），其中hsfl蛋白主要在心肌细胞核内表达，HSP70蛋白主要在心肌细胞浆内表达。结论急性心肌梗死猝死者心肌细胞内hsfl和HSPT0可能共同参与了急性心肌梗死的病理生理过程．这一过程可能是热休克反应的另一调节途径。     

HMGB1在高温致金黄地鼠神经管畸形神经上皮细胞中的表达变化

为了探讨高温致神经管畸形(NTDs)作用的分子机制,为预防人类NTDs的发生提供理论依据,本研究在高温致金黄地鼠NTDs模型的基础上,研究HMGB1在高温致金黄地鼠NTDs神经上皮细胞中的表达变化。将孕鼠随机分为实验组和对照组,分别于水浴处理后8、16、24、40、64 h(相当于胚龄第8、8.5、9、9.5、10.5 d)处死孕鼠,剖腹取鼠胚,制备石蜡切片,应用免疫荧光染色技术检测NTDs发生过程中HMGB1在神经上皮细胞中的表达变化。结果显示:对照组和模型组孕鼠在水浴处理后8、16、24h,HMGB1免疫阳性产物分布于鼠胚神经上皮细胞和周围间充质细胞的胞浆中;水浴后40、64 h,HMGB1表达部位出现了由胞浆向胞核的转移;高温处理后,HMGB1在实验组各期胚胎神经上皮细胞内的表达均比对照组减弱。上述结果提示,HMGB1的表达变化与神经管发育密切相关,其表达降低是高温致神经管畸形发生的重要途径之一。     

热休克因子在乳腺癌细胞株MCF—7热／化疗前后的表达差异及其影响

目的：探讨乳腺癌细胞株MCF-7经热处理及应用阿霉素前后热休克因子（HSF1）的表达差异及基对热估克蛋白（HSP70）以及P-糖蛋白表达的影响。方法：分别给予乳腺癌细胞株MCF-7非致死温度的热冲击43℃1小时、45℃15分钟以及阿霉素处理，继续培养4小时收获细胞，进行免疫细胞化学染色，观察热/化疗前后热休克因子、热休克蛋白以及P-糖蛋白的表达情况。结果：乳腺癌细胞株MCF-7经上述处理后三组细胞的HSF1、HSP70较处理前蛋白表达有明显增高，43℃组、化疗组P-糖蛋白表达较处理前有明显增高，但45℃组P-糖蛋白的表达在热疗前后无明显变化。结论：经热疗或化疗后的乳腺癌细胞株MCF-7中热休克因子表达增高，并能促进热休克蛋白70及P-糖蛋白的表达；热疗可导致化疗耐药的产生。     

鼠巨细胞病毒感染细胞热休克蛋白70与细胞毒T细胞活性的相关性研究

目的体外研究小鼠巨细胞病毒（murine cytomegalovirus,MCMV）感染细胞热休克蛋白70与细胞毒T细胞（CTL）活性相关性。方法体外培养小鼠胚肺细胞,在加入MCMV后16、24、48、72、120h分别收获细胞,用免疫组织化学方法检测HSP70表达情况;CTL活性检测组：体外培养小鼠胚肺细胞,设以下各组：正常细胞对照组,病毒感染组,病毒感染＋放线菌素组,病毒感染＋槲皮素组,病毒感染＋HSP70抗体组,分别在以上时相选用^51Cr释放试验检测CTL活性。结果MCMV感染诱导小鼠胚肺细胞表达HSP70,与正常细胞对照组比较,各时相HSP70表达强度差异均具有显著性,其中16h始升高,120h表达最强。病毒感染组：CTL活性升高,与其它各组比较,各时相差异均具有显著性意义,自16h始升高,120h达最高;放线菌素组、槲皮素组、HSP70抗体组,CTL活性均无显著性差异,自16h始升高,120h达最高,与正常细胞对照组比较,CTL活性均升高。结论MCMV感染细胞热休克蛋白70与CTL的活性具有一定相关性。     

汉坦病毒感染乳鼠脑组织中HSP70与病毒抗原关系

目的：探讨汉坦病毒（HTNV）感染诱导乳鼠脑组织热休克蛋白（HSPs)表达及热休克蛋白与病毒蛋白的相互关系。方法：采用免疫荧光法检测实验性乳鼠感染脑组织中病毒抗原及HSP70的表达，用ELISA、免疫印迹、免疫共沉淀方法分析病毒抗原和HSP70的表达。结果：HTNV感染诱导了乳鼠脑组织神经细胞高表达HSP70，组织中HTNV－NP与HSP70关系密切，免疫共沉淀结果显示HSP70和HTNV－NP结合形成复合物。结论：HTNV实验生乳鼠感染脑组织中高表达的HSP70与病毒核心抗原（HTNV－NP）形成非共价复合物，该复合物可能在病毒感染复制及宿主免疫反应中发挥作用。其潜在的应用价值是在肾综合征出血热的预防和治疗中利用该复合物制备特异性T淋巴细胞疫苗。     

Expressions of HSP70 and HSP27 in hepatocellular carcinoma

The heat shock proteins (HSPs) are ubiquitous molecules induced in cells exposed to various stress conditions, including carcinogenesis. The HSP70 and HSP27 among HSPs are of special relevance in human cancer inhibiting apoptosis. The aim of this study is to investigate the expressions of HSP70 and HSP27 in hepatocellular carcinoma (HCC) in association to tumor cell proliferation and apoptosis. We examined the expressions of HSP70 and HSP27 by immunohistochemical staining in 71 cases of HCC, and then related their expressions to clinicopathologic parameters and expressions of p53, Ki-67 and Apotag. HSP70 and HSP27 were frequently stained in the cytoplasm and nuclei of tumor cells, but not in the non-neoplastic hepatocytes. Immunoreactivities of HSP70 and HSP27 were observed in 56.3% and 61.9% of HCCs, respectively. HSP70 immunoreactivity correlated with high Ki-67 labeling indices (LIs) (p=0.0159), large tumor size (p=0.0129), presence of portal vein invasion (p=0.0231), and high tumor stage (p=0.0392). HSP27 immunoreactivity significantly related with the subgroup of HBV-associated HCCs (p=0.0003), but not with the others. Both HSP70 and HSP27 immunoreactivities showed no relation to Apotag LIs or p53 immunoreactivity. In conclusion, expressions of HSP70 and HSP27 may play an important role in hepatocarcinogenesis, and especially HSP70 showed a close relationship to the pathological parameters associated with tumor progression and high Ki-67 LIs. Our results could be additional evidence that HSP70 expressions can contribute to not only hepatocarcinogenesis but also tumor progression by promoting tumor cell proliferation.     

Heat shock proteins HSP25, HSP60, HSP72, HSP73 in isoosmotic cortex and hyperosmotic medulla of rat kidney

The distribution of heat shock proteins (HSP) HSP60, HSP73, HSP72 and HSP25 in the isoosmotic cortex and the hyperosmotic medulla of the rat kidney was investigated using Western blot analysis and immunohistochemistry. HSP73 was homogeneously distributed throughout the whole kidney. The level of HSP60 was high in the renal cortex and low in the medulla. HSP25 and HSP72 were present in large amounts in the medulla. Only low levels of HSP25 and almost undetectable amounts of HSP72 were found in the cortex. HSP25 exists in one nonphosphorylated and several phosphorylated isoforms. Western blot analysis preceded by isoelectric focussing showed that HSP25 predominates in its nonphosphorylated form in the outer medulla but in its phosphorylated form in cortex and inner medulla. Although this intrarenal distribution pattern was not changed during prolonged anaesthesia (thiobutabarbital sodium), a shift from the nonphosphorylated to the phosphorylated isoforms of HSP25 occurred in the medulla. The characteristic intrarenal distribution of the constitutively expressed HSPs (HSP73, HSP60, HSP25) may reflect different states of metabolic activity in the isoosmotic (cortex) and hyperosmotic (medulla) zones of the kidney. The high content of inducible HSP72 in the medulla most likely is a consequence of the osmotic stress imposed upon the cells by the high urea and salt concentrations in the hyperosmotic medullary environment.     

HSP60, HSP70 and HSP90 from Trichinella spiralis as targets of humoral immune response in rats

This study identifies three heat shock proteins (HSPs) using purified preparations from Trichinella spiralis larvae. The proteins: HSP60, HSP70 and HSP90 were found to be targets of the humoral immune response in rats. Three approaches were adopted to obtain T. spiralis HSP-enriched material and/or to purify HSPs to homogeneity. The former product was prepared using affinity chromatography on gelatin sepharose and elution with ATP. Pure 90 kDa-protein was isolated from parasite extract by sequential DEAE (A50) column chromatograpy and preparative electrophoresis. Immunoblot analysis using monoclonal antibodies to HSP60, HSP70 and HSP90 detected the HSP60 and HSP70 in the affinity-purified product and HSP90 in the product obtained by sequential anionic chromatography and preparative electrophoresis. Finally, the reactivy of preimmune, T. spiralis immune and irrelevant immune rat sera on immunoblots were also examined. Only sera taken from infected rats at time-points after day 7 following the first infection exhibited activity against 60, 70 and 90 kDa proteins on blots. The fact that the serum antibodies were anti-HSP was established by immunoadsorption of HSPs to microtiter plates coated with anti-HSP60, anti-HSP70, or anti-HSP90 and using rat sera, positive on blots, to also give positive scores by continued enzyme-linked immunosorbent assay.     

Expression of heat shock proteins in salivary gland tumors. Immunohistochemical study of HSP27, HSP70, HSP90, and HSP110: apropos of 50 cases

Heat shock proteins (HSPs) are known to be increased in response to biological stress. Recently some authors described their presence in tumors. Our immunohistochemical investigations revealed the expression of HSP27, HSP70, HSP90 and HSP110 in most of benign tumors of salivary glands (33 cases). In the malignant tumors, the reaction was immunopositive for HSP70 and HSP90 in 13/17 cases; but HSP27 and HSP110 were only expressed in 5/17 cases. In conclusion HSPs were expressed less in malignant than in benign cells. These results suggest that the loss of some HSPs may be a possible sign of malignancy.     

Expression of stress proteins HSP 72 & HSP 32 in response to endotoxemia

Pretreatment with heat decreases mortality and acute lung injury in the rat septic shock model, presumably by the production of heat shock proteins (HSP). However, endotoxin, a severe cell stresser, has not been shown to induce HSP 70. We investigated the effects of severe endotoxemia on the expression of specific protective stress proteins, including HSP 72 (inducible HSP 70), HSP 32 (heme oxygenase-1), and HSP 90. Fifteen rats received intravenously either 3 mg/kg of endotoxin (E. coli O127:B8 lipopolysaccharide, LPS) (n=9) or saline (n=6). Two hr later the spleen was removed and splenocytes were separated into three groups and analyzed for specific HSP by Western blot. In Group 1, both endotoxin-treated and saline-treated splenocytes were incubated for 3 hr at 37 degrees C. In Group 2, the splenocytes were washed twice, then heat shocked for 30 min at 42 degrees C and subsequently incubated for 2.5 hr at 37 degrees C. In Group 3, splenocytes were washed twice, then incubated for 3.0 hr at 37 degrees C. HSP 90 & HSP 70c (constitutive) were present in all groups. Consistent with observations by others, HSP 72 was not induced in Group 1. HSP 72 was induced in both the saline-treated and endotoxin-treated splenocytes after heating (Group 2). However, in the absence of heat stress, HSP 72 was present in endotoxin-treated but not in saline-treated splenocytes after incubation (Group 3). Conversely, HSP 32, while present in Group 1 splenocytes, was not detected in the endotoxin-treated splenocytes of Group 2 and Group 3, but was present in the saline-treated cells. In conclusion, endotoxemic shock results in induction of HSP 72 and depletion of HSP 32, but only after the cells have been washed and further incubated.     

Molecular variation of human HSP90alpha and HSP90beta genes in Caucasians

Understanding DNA variation within the human genome is fundamental to the identification and interpretation of genetic components underlying complex traits and diseases. Despite their role in many crucial cellular pathways and their reported involvement in many complex diseases no data are available on the molecular variability of the genes coding for Heat Shock Proteins 90Kda (HSP90). Towards this purpose we have used DHPLC methodology to survey, a sample of Caucasians for genetic polymorphisms in the exons and exon-flanking regions of the expressed genes of human HSP90 gene families, HSP90alpha (HSPCAL4, 14q31.3) and HSP90beta (HSPCB, 6p12). A total of 18 and 11 variants were found in the HSP90-alpha and -beta genes respectively, providing an initial view of human genetic variation in these important genes. Only three of the observed mutations altered the genic product. Interestingly, one of the variations observed was a missense mutation leading to the impairment of the hsp90alpha protein.     

Immunostimulatory properties of the Leishmania infantum heat shock proteins HSP70 and HSP83

Emerging evidence indicates that the heat shock proteins (HSPs), a set of highly evolutionary conserved proteins, are playing essential roles in both normal processes of the immune system and specific immune responses. In a previous work, we demonstrated that the Leishmania infantum HSP70 possesses remarkable immunostimulatory properties. In the present work, we have extended the study to another HSP from this parasite, the HSP83. We show that this protein also has an adjuvant effect to an accompanying protein by stimulation of the humoral response when both proteins are fused and co-administered to BALBjc mice. The analysis of the IgG isotypes, IgG1 and IgG2a, indicated that the immunisations with the Leishmania HSPs, mainly the HSP70, potentiate a Thl-type response. It was found that the amino-terminal domain of the HSP70, the most evolutionary conserved region of the molecule, maintains the ability to stimulate the humoral response, whereas the carboxyl-terminal domain does not have a similar effect. Unexpectedly, we found that the L. infantum HSP70 and HSP83 recombinant proteins stimulated the proliferation of spleen cells from unprimed BALB/c mice. Remarkably, this proliferation was abolished either by thermal denaturing of the proteins or by using specific antibodies. The use of the T-cell inhibitor cyclosporin A in the splenocytes proliferation assays suggested that both T- and non-T-cells are stimulated by the Leishmania HSPs. These findings may be relevant for therapeutic and prophylactic applications.     

Intracellular localization of HSP73 and HSP90 in rat kidneys with acute lysosomal thesaurismosis

We previously reported that HSP73 and HSP90, major chaperone proteins, accumulated within lysosomes of proximal tubular epithelial cells in rat kidneys with acute gentamicin nephropathy. In this study, we observed serial localization of HSP73 and HSP90 in rat kidneys with acute lysosomal thesaurismosis. Sprague-Dawley rats received poly-D-glutamic acid (PDGA) (250 mg/kg per day) for 3 days, and developed acute lysosomal thesaurismosis of proximal tubular epithelial cells. The intracellular localization of HSP73 and HSP90 was examined by electron microscopy. We also compared the results with those of a non-chaperone protein, a renal isoform of argininosuccinate synthetase, which is an abundant enzyme in proximal tubular epithelial cells. After the PDGA exposure, HSP73 and HSP90 accumulated within enlarged lysosomes of proximal tubular epithelial cells. These accumulations started to appear from day 4 after the first PDGA administration, enlarged in size until day 14, and continued until day 19. Argininosuccinate synthetase also accumulated within the lysosomes, but the magnitude of this lysosomal accumulation was less than those of HSP73 and HSP90. Our findings demonstrated that HSP73 and HSP90 chaperone proteins specifically accumulated within lysosomes of proximal tubular epithelial cells during the course of PDGA-induced acute lysosomal thesaurismosis.