激光佐治慢性皮肤溃疡疗效观察及诱导HSP70表达研究

目的:探讨激光治疗慢性皮肤溃疡的疗效及诱导创面组织热休克蛋白70(heat shock prote in 70,HSP70)表达情况。方法:将64例84处慢性皮肤溃疡创面随机分为传统治疗组和激光治疗组,激光治疗组在传统治疗基础上加用激光治疗,所有创面在治疗三周进行疗效判定;同期随机切取5例创面组织,并设正常皮肤为对照,行组织切片HSP70免疫组化染色,计数HSP70阳性细胞数并检测阳性细胞灰度值进行统计学分析;设计引物行RT-PCR检测HSP70mRNA表达情况。结果:两组慢性皮肤溃疡患者经相应治疗均较治疗前明显改善,但激光治疗组创面的治愈率和总有效率明显高于传统治疗组(P<0.05或P<0.01);免疫组化染色显示,正常皮肤和慢性溃疡组织中未见明显HSP70阳性表达细胞,激光治疗后三周后可见较多量的HSP70表达阳性细胞,其阳性细胞计数明显高于其它两组(P<0.01),细胞信号灰度表达明显低于其它两组(P<0.05),而传统治疗组与正常对照组无显著差异;RT-PCR结果发现激光治疗组组织中扩增到一条特异性泳带,大小约268 bp,正常皮肤和传统治疗组组织中未扩增到明显HSP70mRNA基因片段。结论:激光佐治慢性皮肤溃疡具有良好临床疗效,可能与激活创面细胞的热休克内源性保护机制而发挥抗感染和促进愈合作用有关。     

热休克蛋白的生物学作用和转录水平调控研究进展(英文)

热休克蛋白 (HSP)具有广泛的生物学功能 ,其表达方式有两种 :一种是诱导性表达 ,即当生物、细胞受到刺激时才进行表达 ;另一种是组成性表达 ,即在生物、细胞的正常生活、代谢过程中表达。HSP的这两种表达方式意味着HSP基因表达的调控方式和机理不同。本文简要介绍了热休克因子(HSF)的种类、结构及调控HSP基因表达的机理。HSF通过以下 4个步骤调节HSP基因表达 :( 1 )HSF由单体形式变成磷酸化的三聚体形式被激活 ;( 2 )三聚体形式的HSF与HSP基因的热休克元件(HSE)上相邻排列的 3个 5′ GAA 3′结合 ;( 3)与HSF结合后 ,HSE的活化域暴露 ,HSP基因转录 ;( 4 )HSP的mRNA 5′端前导区的特异结构适合于核糖体快速结合和高效翻译。不同生物体内的HSF作用有一定差异 ,功能较为明确的有 :( 1 )对应激信号敏感的HSF1 ;( 2 )对应激信号不敏感 ,对生长、发育、分化信号敏感的HSF2 ;( 3)起抑制HSP基因转录作用的HSF4。还有一些HSF(如HSF3)的作用机制较复杂 ,有待深入研究。此外 ,本文也简要介绍了HSP在衰老、免疫应答、细胞生存和凋亡平衡等中的作用 ,对了解和认识生物生长、发育、衰老、保护、免疫应答及细胞生存和凋亡平衡的分子机制有一定帮助。     

Expression of HSP72 in the gastric mucosa is regulated by gastric acid in rats-correlation of HSP72 expression with mucosal protection

BACKGROUND AND AIM: The real mechanism of adaptive cytoprotection in the gastric mucosa is not well established. In the present study, we investigated the effect of acid suppressing agents on a 72-kDa heat shock protein (HSP72) expression, which is known as endogenous cytoprotective factor, in the gastric mucosa. Also, the association of gastric mucosal protective function against HCl-challenge was compared between HSP72-induced and -reduced group. MATERIALS AND METHODS: Expression of HSP72 was measured by Western blotting in the gastric mucosa before and after administration of famotidine or omeprazole. The gastric mucosal protective function against 0.6 N HCl was compared between control group and HSP72-reduced group. Also, the effect of increased expression of gastric HSP72 by additional administration of zinc sulfate or zinc L-carnosine, which is known as HSP72-inducer, on mucosal protective function was studied. RESULTS: HSP72 expression in the gastric mucosa was reduced by acid suppressing agents. The lowest expression level of HSP72 was observed 12 h (famotidine, H2-receptor antagonist) or 48 h (omeprazole, proton pump inhibitor) after administration. The gastric mucosal protective ability against 0.6 N HCl was also reduced when HSP72 expression was decreased by famotidine or omeprazole. This phenomenon was reversed by HSP72 induction by additional administration of zinc derivatives. CONCLUSION: Our results might indicate that the expression of HSP72 in the gastric mucosa is physiologically regulated by gastric acid, and that HSP72 induction could be important in view of mucosal protection especially when HSP72 expression is reduced by administration of acid suppressing agents such as proton pump inhibitor or H2 receptor antagonist.     

Lymphocyte HSP72 following exercise in hyperthermic runners: The effect of temperature

Heat shock protein 72 (HSP72) is the most inducible HSP, but is not always increased in lymphocytes following exercise. This field study examined whether lymphocyte HSP72 was increased in hyperthermic (T_rec>39.0 °C) male athletes following a 14 km competitive race in cool conditions (ambient temperature 11.2 °C). A comparison was also made between control runners (n=7) and those treated for exertional heat illness (n=9). Lymphocyte HSP72 was not increased in control runners immediately post- compared with pre-race, and there was no difference between both groups of runners. A second study of the race (ambient temperature 14.6 °C) found that lymphocyte HSP72 in control (n=7) and treated (n=9) athletes was higher 2 days post- compared with immediately post-race (p<0.01) and these increases were correlated with post-exercise T_rec (p<0.05).     

低浓度苯并[a]芘诱导赤子爱胜蚓HSP70和HSP90转录上调研究

为寻找土壤低浓度多环芳烃污染分子生物标记物,采用了抑制消减双杂交的方法构建了赤子爱胜蚓在苯并[a]芘（BaP）人工土壤污染胁迫下的差异表达cDNA文库,经测序和基因比对分析后,在上调文库中分别发现2个与热休克蛋白HSP70和1个与HSP90显著匹配的cDNA克隆.经定量PCR验证了0。1 mg·kg^-1和1。0 mg·kg^-1 BaP对赤子爱胜蚓HSP70和HSP90的诱导作用,表明这两个新克隆到的赤子爱胜蚓热休克蛋白基因可作为土壤污染监测的备选分子生物标记物.     

Heat shock protein 72 associated with CD44v6 in human colonic adenocarcinoma

CD44v6 is a splice variant of CD44 (CD44v), probably promoting cancer cell adherence to vascular endothelium and base membranes and enhancing the invasion and metastasis of colonic carcinomas. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to be overexpressed in epithelial carcinoma cells. There may be a possible association between the expression of HSP72 and CD44v6 during the growth and progression of colonic carcinoma cells. The aim of the study was to investigate the interaction between heat shock protein 72 and CD44v6 in human colonic carcinomas. The localization of HSP72 and CD44v6 in human colonic carcinomas was determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and CD44v6 in colonic carcinoma cells was analyzed by immunoprecipitation and Western immunoblots. Our results revealed that colonic carcinoma synchronously co-expressed higher levels of HSP72 and CD44v6 than that in adjacent normal colonic tissues. HSP72 and CD44v6 were mainly immunolocalized in the cytoplasm, and also immunolabelled on the cell membrane. Based on immunoprecipitation and Western immunoblots, we found that HSP72 was associated with CD44v6 precursor fragments in human colonic carcinoma cells. The interaction between HSP72 and CD44v6 in human colonic carcinoma cells may contribute to study the pathogenesis and immunotherapy of colonic carcinoma.     

Restriction fragment length polymorphisms at the heat shock protein HSP70 gene(s) in pigs*

Pigs from a population consisting of eight US breeds or strains and three Chinese breeds were examined by restriction fragment length polymorphism (RFLP) analysis of the heat shock protein HSP70 gene(s). Limited polymorphisms with PstI and PvuII restriction enzymes were observed, but there were no polymorphisms with BomIII and BglI.     

热休克反应中小鼠心脏HSP70表达的免疫组织化学研究

用免疫组织化学方法研究了小鼠心脏在不同温度（40、41、44、46℃）热休克处理后,各恢复期（2、4、8、12、24小时）HSP70的表达。结果表明,（1）44、46℃热处理能诱导心肌细胞合成HSP70,以46℃为多（P＜0．01）,且于恢复期4－8小时为合成高峰（P＜0．01）。（2）阳性免疫反应定位于心肌细胞质中,核呈阴性反应。提示了心脏有较强的耐热能力。     

Temperature-dependent increase in the DNA-binding activity of a heat shock factor in an extract of tobacco cultured cells

The DNA-binding activity of a tobacco heat shock factor (HSF) was induced by heat treatment (37–40 °C) of a cell-free extract that contained extra-nuclear fraction, but not in an extract of isolated nuclei. These observations suggest that an inactive form of HSF can directly recognize and transduce the heat shock signal and that such transduction requires components of the extranuclear fraction. Addition of ATP or of most other nucleoside triphosphates reduced the binding of the HSF to the heat shock element (HSE) in the same extract, and removal of ATP by dialysis from the extract restored the ability of the HSF to bind to DNA. The restored activity of the HSF could be eliminated again by a second addition of ATP. Our observations provide the first example of the involvement of ATP in the regulation of the reversible changes in HSF that control its ability to bind to HSEs in a cell-free extract.Abbreviations AMP-PNP adenylyl imidodiphosphate - GUS -glucuronidase - HSE heat shock element - HSF heat shock factor     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Extracellular HspBP1 and Hsp72 synergistically activate epidermal growth factor receptor

Background information. Heat‐inducible Hsp72 is the founding member of the Hsp70 (heat shock proteins of 70 kDa) family of molecular chaperones. It is localized primarily in cytoplasm and nucleus but is also found extracellularly. The source of e‐Hsp72 (extracellular Hsp72) is not precisely identified and may not be the same in every situation. A number of studies demonstrated that e‐Hsp72 plays an important role in cell survival, tumour rejection and immune response. However, currently little is known about regulation of e‐Hsp72 function. In cells, Hsp72 is controlled by co‐chaperones. An abundant co‐chaperone, HspBP1 (Hsp72‐binding protein 1) was found extracellularly in the serum. In the present study we analysed the secretion and function of e‐HspBP1 (extracellular HspBP1). Results. A431 human squamous carcinoma cells accumulated Hsp72 and HspBP1 in chromogranin A‐positive granules following heat stress or in the presence of U73122, an inhibitor of phospholipase C. Following these treatments, A431 cells also increased the secretion of both proteins into the culture medium. The secreted e‐Hsp72 and e‐HspBP1 were co‐immunoprecipitated from the conditioned medium. Purified recombinant HspBP1 augmented e‐Hsp72‐mediated phosphorylation of EGFR (epidermal growth factor receptor) and its down‐stream targets, ERK1 (extracellular signal‐regulated kinase 1) and ERK2 in a concentration‐dependent manner. Finally, a HspBP1 N‐terminal domain deletion mutant and boiled recombinant HspBP1 did not affect the e‐Hsp72‐mediated activity. Conclusions. Heat stress and PLC (phospholipase C) inhibition result in the enhanced secretion of both Hsp72 and HspBP1. In an extracellular environment, the two chaperones interact both physically and functionally, leading to the activation of th EGFR—ERK1/2 signalling pathway. However, the magnitude of EGFR activation depends on the e‐HspBP1/e‐Hsp72 ratio in the medium. Extracellular chaperone‐mediated activation of EGFR can provide a survival advantage to cells under heat shock and other stresses.     

黄粉虫热休克蛋白70基因的克隆、序列分析与表达（英文）

为给黄粉虫Tenebrio molitor抗逆机理研究提供理论依据, 本研究采用PCR和RACE法从黄粉虫幼虫中克隆出一个热休克蛋白70基因Tmhsp70, 并运用半定量RT-PCR法检测其在黄粉虫不同发育阶段的mRNA表达水平。结果表明： 克隆出的Tmhsp70 序列全长2 282 bp, 具有一个富含A的115 bp 5′ 非翻译区和一个1 935 bp的开放阅读框及一个富含A、 T的232 bp 3′-非翻译区。5′-非翻译区含有7个热休克元件nGAAn, 3′-非翻译区末端有长22 bp的Poly(A)尾。Tmhsp70编码的黄粉虫热休克蛋白（TmHSP70）具有3个典型的HSP70特征基序（IDLGTTYS, IFDLGGGTFDVSIL和IVLVGGSTRIPKIQQ）和1个胞质HSP70末端特征基序（EEVD）, 无信号肽和跨膜区域, 包含2个主要的结构域, 即： N-端42 kDa的高度保守ATPase功能域和C-端18 kDa的保守多肽结合功能域。ATPase功能域的三级结构由2个大球形亚功能域组成, 具有1个核苷酸结合中心; 多肽结合功能域形成1个双层4股β-折叠片样的三明治结构和2个α-螺旋, 内含1个多肽结合通道。此外, 黄粉虫Tmhsp70 mRNA的表达具有热激诱导和发育调控的特征。半定量RT-PCR分析表明, 42℃热激1 h的黄粉虫各发育阶段Tmhsp70 mRNA的表达量上升了1.4~26.9倍。25℃下1日龄黄粉虫蛹中的Tmhsp70 mRNA 表达量要高于其余各发育阶段的累积表达量; 42℃热激1 h 后90日龄幼虫中的Tmhsp70 mRNA 表达量最丰富, 既高于30日龄和60日龄幼虫中的累积表达量, 也高于15日龄和30日龄成虫中的累积表达量。这些结果为进一步研究黄粉虫热休克蛋白的结构、 功能和表达调控及其与抗逆性的关系奠定了基础。     

Cryptic Expression of the 70-kDa Heat Shock Protein,hsp72, in Gerbil Hippocampus after Transient Ischemia

The 70 kDa heat shock protein, hsp72, is known to be induced following transient global ischemia in brain, as detected by immunocytochemistry and in situ hybridization techniques. However, while hsp72 mRNA is expressed rapidly following postischemic recirculation, immunocytochemistry fails to detect hsp72 protein for many hours after such insults, even in cell populations that readily express Fos and other proteins encoded by ischemia-induced mRNAs. In the present study, hsp72 expression in gerbil hippocampus was compared by immunocytochemistry and immunoblot methods at several intervals following 10 min ischemia. As established in previous studies, hsp72 immunoreactivity remained undetectable in postischemic neurons at 6 h following such insults. In contrast, immunoblots of dissected gerbil hippocampus demonstrated nearly maximal accumulation of hsp72 at this time point. These results indicate that the protein is present, but cryptic to detection in perfusion-fixed sections, during early recirculation. The constitutively expressed heat shock cognate protein, hsc70, did not show significant changes in level or distribution by either method, except for a decrease in CA1 staining at 48 h. These results confirm that hsp72 rapidly accumulates to high levels in postischemic hippocampus, and suggest that further studies of its subcellular localization during this interval may offer insight into its functional role as a component of the stress response in neurons after such insults.     

GmHSFA1基因克隆及其过量表达提高转基因大豆的耐热性

热激转录因子在调节植物对逆境胁迫应答和热激蛋白基因表达方面起重要作用。采用生物信息学和比较基因组学方法结合RACE技术从大豆基因组中克隆到一个新的热激转录因子基因GmHsfA1, 其cDNA全长1 781 bp, 包含1个1 533 bp的开放阅读框, 编码含有510个氨基酸残基的蛋白质(GenBank登录号为AY458843)。与其他转录因子的分子结构相似,GmHSFA1也含有4个典型的结构功能域—DNA结合域、寡聚域、核定位信号和C端激活域。BLAST分析表明, GmHSFA1与其同源性最高的番茄热激转录因子LpHSFA1之间的氨基酸序列相似性为52.46%。RT-PCR、Northern和遗传转化结果显示: 1)GmHsfA1在大豆的不同组织中呈现组成型表达模式; 2)常温下转基因大豆植株的GmHsfA1表达水平明显高于非转基因对照; 3)GmHsfA1的过量表达激活了转基因大豆植株中热激蛋白基因GmHsp22在非诱导条件下的转录, 并加强了高温胁迫下另2个热激蛋白基因GmHsp23和GmHsp70的表达; 4)转GmHsfA1大豆植株的耐热温度(达52℃)明显高于非转基因植株。上述结果说明, GmHsfA1的过量表达激活或促进其下游3个热激蛋白基因的转录或表达, 明显提高了转基因大豆植株的耐热能力。