外源性热休克蛋白70基因对心肌细胞损伤的保护作用

目的：探讨热休克蛋白70（HSP70）基因转染对心肌细胞急性实验性缺氧／复氧损伤的保护作用及其机制。方法：进行大鼠乳鼠原代心肌细胞培养,随机分为4组,对照组、缺氧／复氧（A／R）组、热休克处理后缺氧／复氧（A／R＋HS）组和pCDNAHSP70质粒转导后缺氧／复氧（A／R＋pCDNA HSP70）组。用脂质体包裹pCDNAHSP70质粒,并转导入心肌细胞内。反转录-聚合酶链反应（RT-PCR）检测HSP70mRNA表达,Westernblot检测HSP70蛋白表达。以心肌细胞存活率（MTT法）、培养液中乳酸脱氢酶（I．DH）活性、心肌细胞超微结构改变（透射电镜）及Ca^2＋负荷来检测心肌细胞损伤程度。结果：与A／R组相比,A／R＋HS组和A／R’pCDNAHSP7。组细胞存活率显著提高;LDH活性明显降低;细胞超微结构明显改善;心肌细胞Ca^2＋负荷明显减轻。A／R组HSP70mRNA和蛋白含量均较A／R＋HS组和A／R＋pCDNAHSP70组显著减低（P〈0．01）,而A／R＋pCDNA HSP70组的含量与A／R＋HS组相比显著增多（P〈0．01）。结论：通过基因转染使HSP70基因高表达能对抗缺氧／复氧对心肌细胞的损伤,这一作用与其能抗细胞Ca^2＋负荷有关。     

Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells

Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic acid-induced small intestinal apoptosis, a hallmark of acetylsalicylic acid-induced enteropathy.     

热休克蛋白90α、70、27mRNA在急性白血病细胞中表达的研究

运用RNA分子杂交的方法,观察了热休克蛋白(heat shock protein,HSP)90α、70、27在22例白血病病人细胞,正常血细胞及K562红白血病细胞系中的表达。结果表明:16例白血病病人中,4例急性淋巴细胞白血病(ALL),6例急性非淋巴白血病(ANLL)、1例慢性粒细胞白血病急变期(慢粒急变)和2例骨髓增生异常综合征(MDS)血细胞呈现HSP27高水平表达,较正常血细胞显著增多,ALL(5例)与ANLL(7例)白血病细胞HSP27表达水平无明显改变。检测了7例白血病细胞HSP70的表达水平,除1例ALL及1例MDS明显升高外,其余5例(包括1例ALL,3例ANLL和1例慢粒急变)显著低于正常血细胞。17例白血病病人细胞(包括9例ANLL、5例ALL、2例慢粒急变和1例MDS)HSP90α表达水平均升高,明显高于正常。结果提示:白血病细胞HSP90α表达水平的升高可能与白血病细胞活跃异常增殖有关,而HSP27基因的高表达可能不是某种急性白血病的特殊标志。     

解脲脲原体热休克蛋白HSP70与男性不育的相关研究

支原体是引起不育的致病菌之一。解脲脲原体通过改变精液参数、影响精子运动方式、产生抗精子抗体、导致精子凋亡数增加而引起男性不育。解脲脲原体热休克蛋白与男性不育的关系主要在于热休克蛋白70(HSP70)可以抑制解脲脲原体细胞凋亡。     

热休克蛋白70在大鼠黑质多巴胺神经元损伤中的表达

目的 :探讨热休克蛋白 70 (HSP70 )在大鼠黑质多巴胺 (DA)神经元损伤中的表达以及在帕金森病 (PD)诊断中的意义。方法 :将 48只大鼠随机分为黑质DA神经元损毁组 (PD组 )和对照组各 2 4只。PD组注射 6 羟多巴胺 (6 OHDA)损毁大鼠黑质DA神经元 ,对照组仅注射 6 OHDA溶媒。于注射后 1、7、14及 2 1d采用免疫组织化学、尼氏染色、电镜手段动态观察HSP70在损毁的DA神经元中的表达以及DA神经元形态学变化。结果 :在 6 O HDA损毁黑质 1～ 2 1d ,对照组黑质HSP70表达和尼氏细胞计数差异无显著性 (P >0 .0 5 )。PD组HSP70表达在 1d最高 ,7d锐减 ,14和 2 1d则逐渐减少 ,分别为 2 5 %、74%、87%及 88% ;尼氏细胞计数在 4个时间点分别减少1%、13%、35 %及 48% ;超微结构损伤程度呈进行性加重。结论 :PD渐进性发病具有其形态学基础 ,HSP70可作为DA神经元存活的指标和早期诊断PD的指标。     

热休克及化疗刺激对人白血病细胞K562中HSP70 mRNA及MDR1 mRNA表达的影响

为了研究热休克、Adriamycin（ADM）化疗应激刺激下热休克蛋白70（HSP70）、多药耐药基因MDRl的表达情况，对K562细胞予以43℃高温热休克和（或）加以ADM化疗，然后采用免疫组织化学方法检测HSP70的表达，MTT法检测K562细胞的生长抑制率，RT—PCR检测HSP70 mRNA和MDRl mRNA的表达，流式细胞术检测P-糖蛋白（P—gP）的表达。结果显示：①HSP70蛋白的合成在43℃热作用后恢复37℃孵育2小时达到高峰并聚集在胞核和核仁内，呈“核内迁移”现象；3小时后HSP70表达开始降低，5小时后HSP70的合成逐渐降低至正常，并恢复以胞浆表达为主。②K562细胞在43℃高温热休克刺激下HSP70mRNA表达增加，随着37℃孵育时间的延长表达量有增多趋势并持续一段时间，MDRlmRNA的表达与之相似，二者在37℃孵育2小时分别增加4倍和5．8倍。③ADM化疗刺激后细胞内P—gP表达增加；ADM化疗、43℃热休克刺激后再加ADM化疗，K562细胞中HSP70 mRNA、MDRl mRNA表达均上调，且43℃热休克刺激处理后再加入ADM作用二者的表达要高于单加ADM化疗组。结论：热休克和ADM化疗刺激的K562细胞中HSP70和MDRl表达增加，有利于维护肿瘤细胞的稳定性，HSP70可能与耐药有关。     

放线菌素D诱导卵巢癌细胞凋亡中热休克蛋白(HSP)的作用的研究

目的探讨热休克蛋白(heat shock protein,HSP)在放线菌素D(actinomycin D,Act-D)诱导卵巢癌细胞A2780凋亡中的作用,为进一步探讨热休克蛋白抗凋亡机制及卵巢癌生物治疗提供实验依据。方法体外培养的卵巢癌细胞A2780于RPMI1640,10%(v/v)胎牛血清,5%(v/v)CO_2孵育,取指数生长期细胞1×10~5/ml接种反应板24h,分为非热休克组(NHS)和热休克组(HS),两组分别由不同浓度(5,10,20μg/ml)Act-D诱导,作用4h。采用脱氧核苷酸末端转移酶介导的dUTP缺口末端标记法(TUNEL)和~3H-TdR掺入试验检测肿瘤细胞凋亡指数(AI)及DNA合成情况。结果HS组比NHS组凋亡指数明显下降(P<0.01);cpm值明显升高(P<0.01),呈现剂量依赖效应。结论热休克反应对卵巢癌细胞A2780经Act-D诱导凋亡具有抑制作用,此作用与Act-D呈剂量依赖效应。     

热休克蛋白90在多发性骨髓瘤患者中的表达及临床意义

目的:检测多发性骨髓瘤(MM)患者骨髓标本中HSP90的表达情况,并探讨其临床意义.方法:利用免疫组化技术检测HSP90在76例MM患者及29例健康供者中的表达;收集临床资料,分析HSP90表达情况与临床指标的相关性.结果:MM患者骨髓标本中HSP90表达阳性患者数明显高于对照组;HSP90表达水平与患者的β2-微球蛋...     

Expression of Heat Shock Protein 70 in Rabbit Liver After Contrast-Enhanced Ultrasound and Radiofrequency Ablation

Heat shock proteins (HSPs) induced by thermal ablation therapy may help presenting tumor antigen to the host immune system and be a valuable adjuvant in the ablation therapy of liver cancer. This paper described our preliminary study on the expression of HSP70 in rabbit liver after contrast-enhanced ultrasound (CEUS) and radiofrequency (RF) ablation. Twenty-five male New Zealand white rabbits were divided into five groups as: control group (n=5), ultrasound group (n=5), CEUS group (n=5), RF group (n=5) and CEUS + RF group (n=5). Clinical ultrasound and RF ablation equipment were used in the present experiment. Sonazoid was used as the contrast agent. All the animals were sacrificed 24 h after the procedure, and HSP70 was detected by immunohistochemistry staining and Western blot analysis. In the groups without RF ablation, there was no evidence of HSP70 expression in the liver tissue of the control group and ultrasound group, whereas positive HSP70 expression was detected in the liver tissue of the CEUS group, with a mean optical density of 0.33. In the RF and CEUS + RF groups, there were cells showing HSP70 expression in the normal liver tissue far from the ablation region. The mean densities of HSP70 expression were 0.31 in the RF group and 0.35 in the CEUS + RF group, respectively. With regard to the distribution of HSP70 expression of the RF and CEUS + RF groups, the marginal areas were stronger than liver tissue 1 cm away from the margin, and the ablated tissues showed no evidence of HSP70 expression. The mean density of HSP70 expression in the marginal areas were 0.47 in the RF group and 0.42 in the CEUS + RF group, respectively. CEUS using Sonazoid may produce HSP70 expression in the normal liver parenchyma after CEUS examination and RF ablation. (E-mail: moriyasu@tokyo-med.ac.jp)     

从K562细胞中提取热休克蛋白GP96及其对人树突状细胞分化和功能的影响

本研究旨在建立从K5 6 2细胞系中提取热休克蛋白GP96的方法 ,进而研究GP96对人树突状细胞分化和功能的影响。采用蛋白分离提纯技术 ,将K5 6 2细胞裂解 ,经饱和硫酸铵沉淀、亲和层析、离子交换层析后提纯出热休克蛋白GP96 ,用SDS PAGE凝胶电泳和Westernblot方法对其进行鉴定。将提纯的GP96加入从正常人外周血单个核细胞诱导培养的树突状细胞 (dendriticcell,DC)中 ,通过流式细胞术检测DC细胞表型变化 ,用MTT法测定混合淋巴细胞反应 (mixedlymphocytereaction ,MLR)。结果表明 :从 1× 10 1 0 K5 6 2细胞中能提纯出热休克蛋白GP96 6 0 - 80 μg ;加入GP96制备的HSP DC ,其细胞表面标志CD83、CD86和HLA DR的表达率比常规培养DC明显升高 ,CD1a表达率降低 (P <0 .0 5 ) ;HSP DC比常规培养DC具有更强的刺激混合淋巴细胞反应的能力 (P <0 .0 5 )。结论 :从K5 6 2细胞中可以提取出热休克蛋白GP96 ;热休克蛋白GP96可以促进树突状细胞分化成熟 ,使其具有更强的刺激同种异体人T淋巴细胞增殖的能力     

谷氨酰胺不同作用时间对单个核细胞热休克蛋白70表达的影响

目的 探讨经内毒素(lipopolysaccharide,LPS)预处理后,谷氨酰胺(glutamine,Gln)不同作用时间对单个核细胞(peripheral blood mononuclear cell,PBMC)热休克蛋白70(HSP70)表达的影响.方法 取健康志愿者新鲜外周血滤过的血盘,用0.9％氯化钠注射液按1∶1稀释血液,Ficoll-泛影酸钠(Ficoll-Hypaque)密度梯度离心法提取PBMC,以LPS刺激PBMC2h后,使用10 mmol/L的Gln分别刺激PBMC 0、0.5、1、2、3h后提取蛋白,采用Western blot方法检测PBMC中HSP70表达情况.结果 PBMC经LPS刺激2h后,予Gln分别作用0、0.5、1、2、3h,结果显示作用1h后,HSP70的表达量最高,与其他时间段比较差异均有统计学意义(P＜0.05),随后表达量减少;作用2、3h后表达量虽有减少,但与作用0.5h比较,差异亦有统计学意义(P＜0.05).结论 早期补充Gln可上调LPS诱导的PBMC中HSP70的表达,Gln发挥作用快速,但作用时间短.     

Correlation of Heat Shock Protein Expression to Gender Difference in Development of Stress-Induced Gastric Mucosal Injury in Rats

Recent studies have indicated that heat shock proteins (HSPs), which function as molecular chaperones, play important roles in cellular responses to stress-related events. However, the gender difference in the expression of HSP in the gastric mucosa remains unclear. In order to understand the mechanism of gender difference in the prevalence or severity of gastric mucosal lesions, the expression level of HSP and the correlation of estrogen to HSP induction in the gastric mucosa were evaluated in this study. The basal expression levels of HSP60 and HSP90 in the gastric mucosa were significantly higher in females than those in males. The gastric ulcer index was significantly higher in male rats compared to female rats observed after 12 h water immersion stress exposure. At this time point, the expression levels of HSP60 and HSP90 in the gastric mucosa were significantly higher in females than those in males. An estrogen-treatment significantly induced the expression of HSP60, HSP70 and HSP90 in the gastric mucosa. Inversely, an ovariectomy dramatically reduced the expression of HSP60, HSP70 and HSP90 in the gastric mucosa. Our results suggested that estrogen might play an important role in gastric mucosal protection with the induction of gastric mucosal HSPs.     

热休克预处理对严重烫伤大鼠胃黏膜氧自由基的作用

目的:观察热休克预处理对严重烫伤大鼠胃黏膜氧自由基及热休克蛋白(HSP)70表达的影响,探讨氧自由基在大鼠烫伤后急性胃黏膜损伤中的作用及热休克预处理对严重烫伤大鼠胃黏膜的保护作用机制。方法:将96只Wistar大鼠随机分为烫伤组(B组,n=48,其中8只大鼠作为正常对照)及热休克预处理后烫伤组(HB组,n=48,其中8只大鼠作为实验对照),观察各时相点两组大鼠胃黏膜损伤指数(UI)、胃黏膜丙二醛(MDA)及胃黏膜细胞SOD活性变化。应用免疫组化染色分析胃黏膜细胞中HSP 70表达变化。结果:大鼠严重烫伤后胃黏膜细胞SOD大量消耗,MDA显著增加,胃黏膜损害明显;热休克预处理能显著减轻大鼠严重烫伤后急性胃黏膜损害。HB组较B组大鼠胃黏膜HSP 70表达水平显著升高(P<0.05),热休克预处理能显著降低胃黏膜细胞SOD的消耗,减少MDA的产生。胃黏膜损伤指数(UI)与MDA呈显著正相关(r=0.695,P<0.01),与HSP 70呈显著负相关(r=-0.794,P<0.01);胃黏膜SOD与MDA呈显著负相关(r=-0.823,P<0.01),而与HSP 70呈显著正相关(r=0.527,P<0.05)。结论:严重烫伤大鼠胃黏膜存在氧自由基损伤;热休克预处理可显著减轻烫伤大鼠胃黏膜氧自由基损伤,保护机制与HSP 70保护抗氧化酶SOD活性密切相关。     

中性粒细胞膜表面与胞内热休克蛋白70含量比值和严重脓毒症患者预后的相关分析

目的:探讨外周血中性粒细胞膜表面与胞内热休克蛋白70(heat shock protein 70,HSP70)含量的比值对严重脓毒症患者病情及预后的影响.方法:采用前瞻性随机对照的临床试验设计,选择上海市浦东新区公利医院急诊科确诊的严重脓毒症患者60例作为研究组,同期40例健康人群作为健康对照组.采用流式细胞术检测入急诊科24 h内研究对象外周血中性粒细胞膜表面与胞内HSP70表达水平,同时在进入急诊室24 h内进行简化急性生理学评分Ⅱ(simplified acute physiology score Ⅱ,SAPSⅡ),采用集束化治疗策略治疗,治疗28 d后判定患者的预后,比较研究组内存活患者(存活亚组)与病死患者(病死亚组)的中性粒细胞膜表面与胞内HSP70含量的比值和SAPSⅡ,并对2组中性粒细胞膜表面与胞内HSP70含量的比值和SAPSⅡ进行相关分析.结果:研究组患者中性粒细胞膜表面与胞内HSP70含量的比值显著高于对照组[(0.76±0.53)比(0.08±0.10)],P＜0.01.研究组60例患者中存活42例,病死18例,存活亚组中性粒细胞膜表面与胞内HSP70含量的比值显著低于病死亚组[(0.68±0.13)比(0.83±0.18)],P＜0.05;存活亚组SAPSⅡ显著低于病死亚组[(48.30±8.15)比(68.30±18.58)],P＜0.05.相关性分析显示,中性粒细胞膜表面与胞内HSP70含量的比值与SAPSⅡ呈显著正相关(r=0.295,P=0.01).结论:外周血中性粒细胞膜表面与胞内热休克蛋白HSP70含量的比值可反映严重脓毒症患者病情及预后.     

高血压脑梗死与血浆热休克蛋白70抗体水平关系的研究

目的通过测定高血压脑梗死患者血浆热休克蛋白70（HSP70）抗体水平，探讨HSP70抗体与脑梗死损伤程度的关系及其在脑梗死发病机制中的作用。方法100例脑梗死患者经头颅CT或MRI确诊，其中男64例，女36例；高血压者67例，血压正常者33例。选100名社区居民作为对照组，其中男58名，女42名；高血压者35名，血压正常者65名。采用蛋白质免疫印迹-酶联免疫吸附法（Western blot—ELISA）测定血浆HSP70抗体，并进行Logistic回归分析。结果脑梗死组HSP70抗体阳性率明显高于对照组（69％比13％）；脑梗死组高血压者HSP70抗体阳性率明显高于血压正常者（57％比12％），对照组高血压者HSP70抗体阳性率亦高于血压正常者（11％比2％）。上述结果差异均有显著性（P均〈0．05）。结论HSP70抗体增多与高血压脑梗死发病相关，可作为高血压脑缺血时一种分子生物学诊断指标；HSP70抗体表达越强，脑梗死病情越严重。因此，HSP70抗体增高是反映脑组织损伤程度的标志。     

Induction of heat shock protein 70 genes in human lymphocytes during fever therapy

The induction of heat shock protein 70 ( HSP70 ) genes has been studied in peripheral blood mononuclear cells of individuals undergoing fever therapy because of metastatic malignant melanoma. Induction of HSP70 was assessed at the protein level by metabolic labelling or, for the HLA-linked HSP70-1 and HSP70-2 genes, at the RNA level by in situ hybridization. However, de novo expression of HSP70 could be observed during fever (usually above 39°C) in only about half of the cases. No simple threshold model for inducibility of HSP70 in vivo could be applied. The HSP70-1 gene was induced more easily than HSP70-2 . Thus, heat-inducible HSP70 genes, including HLA-linked HSP70 genes, become expressed in human lymphocytes during fever, but not regularly.     

健脾益气中药对消化性溃疡患者HP及HSP60的影响

目的：探讨健脾益气中药对消化性溃疡（PU）患者幽门螺杆菌（HP）清除及热休克蛋白HSP60的影响。方法：临床观察HP清除率，运用斑点印迹技术^[1]及免疫组织化学方法检测血清及胃黏膜HSP60。结果：清除率在中药组与三联组之间无明显差异（P＞0．05，HSP60含量在中药组较三联组显著增高（P＜0．01）。结论：健脾益气中药对HP有清除作用，这种作用可能与免疫反应相关。     

HSP70基因转染对缺氧-再复氧肠上皮细胞生长能力影响的研究

目的 :研究重组腺病毒介导的热休克蛋白 70转染对缺氧再复氧损伤后肠上皮细胞生长能力的影响。方法 :将重组含人全长 HSP70基因的腺病毒转染体外培养的肠上皮细胞株 IEC 6 ,检测转染细胞 HSP70的 m RNA表达水平。 IEC 6细胞经缺氧再复氧处理后 ,对未转染组 ,转染 2 4 h、4 8h及 72 h组细胞的细胞活力、生长曲线、增殖能力进行检测分析。结果 :腺病毒转染组细胞 HSP70 m RNA表达呈阳性。经缺氧再复氧处理后 ,IEC 6转染细胞活力较未转染组活力明显增强 (P<0 .0 1) ,生长能力提高 (P<0 .0 5 ) ,分裂指数增高(P<0 .0 5 )。结论 :重组腺病毒介导的 HSP70转染可增强肠上皮细胞生长及增殖能力 ,从而保护其抵抗缺氧再复氧损伤。