热休克蛋白在子宫内膜异位症中的表达及意义

目的 探讨热休克蛋白27（HSP27）和HSP70在子宫内膜异位症发病中的作用。方法 采用免疫组化链霉菌抗生素蛋白－过氧化物酶染色法（SP法），检测子宫内膜异位症中56例异位内膜与在位内膜（30例）中HSP27和HSP70的表达。采用原位杂交，检测HSP70mRNA的水平，并与正常子宫内膜相比较。结果 免疫组化结果显示，异位内膜组织中HSP27与HSP70均呈高表达，与正常内膜组的表达差异显著（P＜0．01），而且失去其在正常内膜组织中表达的周期性变化。卵巢子宫内膜异位症组（OEM）与子宫腺肌症组（AM）组中，HSP27和HSP70的表达无显著差异（P＞0．05）。原位杂交结果显示，HSP70 mRNA（分别为P＜0．01和P＜0．05）。HSP27与HSP70的表达水平，同OEM的严重性无关。结论 异位内膜组织中的HSP70基因被激活，转录增加。HSP27和HSP70呈高表达，可能在EM的发病中起重要作用。     

Expression of heat shock proteins in classical Hodgkin lymphoma: correlation with apoptotic pathways and prognostic significance

Santón A, García‐Cosío M, Cristóbal E, Pascual A, Muriel A & García‐Laraña J (2011) Histopathology 58, 1072–1080 Expression of heat shock proteins in classical Hodgkin lymphoma: correlation with apoptotic pathways and prognostic significance Aims: Heat shock proteins (HSPs), known to inhibit apoptosis and promote cellular survival, are overexpressed in many tumours. We analysed the expression of relevant HSPs and heat shock factor 1 (HSF1) in classical Hodgkin lymphoma (cHL) and their relationship with caspase signalling pathways and patient outcome. Methods and results: Using tissue microarrays (TMAs), most cases showed strong immunohistochemical expression of HSPs [10, 27, 40, 60, 70, 90, 110, HO1, cell division cycle 37 homolog (CDC37) and HSF1, which points to cHL as a potential candidate to stress‐response inhibitors. Active caspases 3, 8 and 9 were detected in 55.1%, 55.4% and 96.2% of cases although cleaved poly (ADP‐ribose) polymerase (PARP) was observed in only 16.1%, suggesting an improper functioning of apoptosis. Statistical analysis showed associations of HSP70 with active caspase 3 (P = 0.000); HSP40 with active caspase 9 (P = 0.031) and p53 (P = 0.003); HO1 with p53 (P = 0.006) and p21 (P = 0.005); and p53 with p21 (P = 0.015). Conclusions: Correlations between the expression of apoptotic markers and HSPs may suggest a role for the latter in modulating apoptosis in cHL, mainly through the HSP70‐HSP40 system, and in the stabilization of p53. Survival analyses showed that absence of active caspase 8 and HO1 had a negative impact in patient outcome.     

Distribution of heat shock proteins in kidneys of rats after immunosuppressive treatment with cyclosporine A

Cyclosporine A (CsA), a fungal undecapeptide, is the most common immunosuppressive drug used in organ transplantation and auto-immune diseases. However, it has severe side effects mainly on renal structures and functions. Therefore, nephrotoxicity is the major limiting side effect. Heat shock proteins (HSPs) are molecular chaperones, that are induced or expressed at high levels in mammalian cells due to a variety of adverse effects. HSPs have beneficial roles in protein processing and protection against cell injury. In the present study, we examined immunohistochemically levels of expression and localization patterns of various HSPs in rat kidneys after administration of a therapeutic CsA dose during 30 days. After CsA treatment, both constitutive HSP 25 and alpha B-crystallin immunoreactivity became stronger in glomeruli, proximal tubules and collecting ducts. Nuclear translocation of these proteins was detected in renal tubules. HSP 47 was detected in the interstitial space between tubules, vascular smooth muscle and medullary rays. Finally, HSP 72 was induced in the cytoplasm of epithelial cells of proximal and distal tubules, and in the cytoplasm of epithelial cells of Henle limbs and collecting ducts. These data demonstrate that CsA clearly induces increased immunoreactivity of HSPs in defined structures of rat kidneys. These findings suggest that these proteins are functionally involved in the defence against renal cellular damage caused by prolonged drug treatment in rat.     

The role of heat shock proteins in reproduction

Heat shock proteins (HSP) were first identified in cells after exposure to elevated temperature. Subsequently HSP have been identified as a critical component of a very complex and highly conserved cellular defence mechanism to preserve cell survival under adverse environmental conditions. HSP are preferentially expressed in response to an array of insults, including hyperthermia, free oxygen radicals, heavy metals, ethanol, amino acid analogues, inflammation and infection. HSP interact with intracellular polypeptides and prevent their denaturation or incorrect assembly. In addition HSP are also involved in several processes essential for cellular function under physiological conditions. HSP production is enhanced during in-vitro embryo culture and they are among the first proteins produced during mammalian embryo growth. The spontaneous expression of HSP as an essential part of embryo development is well documented and the presence or absence of HSP influences various aspects of reproduction in many species. Finally, HSP are immunodominant antigens of numerous microbial pathogens, e.g. Chlamydia trachomatis, which have been recognized as the main cause of tubal infertility. Many couples with fertility problems have had a previous genital tract infection, have become sensitized to microbial HSP, and a prolonged and asymptomatic infection may trigger immunity to microbial HSP epitopes that are also expressed in man. Antibodies to both bacterial and human HSP are present at high titres in sera and hydrosalpinx fluid of many patients undergoing in-vitro fertilization (IVF). In a mouse in-vitro embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages. Recent studies indicate an association between a previous infection, immunity to HSP and reproductive failure.     

热休克蛋白与抗感染免疫

热休克蛋白(heatshockproteins,HSPs)又称为应激蛋白,由多基因家族及相关基因产物诱导产生,参与高度保守的细胞防御机制.作为细胞内分子,HSPs参与蛋白成熟必要的生化途径,对于正常细胞自稳状态的维持起重要作用.除上述分子伴侣功能外,HSPs还具有另一种重要的生物特性--免疫原性.在感染引起的免疫性疾病的发生和发展中,HSPs具有防止感染应激引起的细胞损害和促进受损细胞恢复等重要作用.作为感染过程中体液和细胞免疫的重要靶目标,HSPs用于感染性疾病的疫苗研究日益受到人们的关注.     

Heat shock proteins in human endometrium throughout the menstrual cycle

Human endometrium is a steroid-sensitive tissue and there isevidence that supports the viewpoint that heat shock proteins(HSP) are implicated in the regulation of steroid function.Therefore, in this study we examined the expression of variousmembers of the heat shock family of proteins in the steroid-responsivehuman endometrium. Western blot analysis revealed that the expressionof HSP90 showed minimal changes throughout the menstrual cycle.When normalized to the amount of HSP90, the expression of HSP27,HSP60 and the constitutive form of heat shock protein 70 (HSC70)increased progressively during the late proliferative and earlysecretory phases, and diminished in the mid- to late secretoryand menstrual phases. In contrast, the inducible form of heatshock protein 70 (HSP70) did not undergo these changes. Thecellular and subcellular localizations of these proteins wereexamined in human endometria by immunohistochemical staining.With the exception of HSP70, which was found primarily in theepithelial cells, the immunoreactivity for other heat shockproteins was found in both the stroraa and the epithelium. Immunoreactivityfor HSP27 was found in the lymphoid aggregates within endometrialstroma, and both HSP27 and HSP90 were found in endothelial cells.The immunoreactive heat shock proteins were found in the nucleiand/or cytoplasm of cells. However, no consistent nuclear versuscytoplasmic staining emerged, and such localization was irrespectiveof the site, the cell type or the phase of the menstrual cycle.Our findings show that endometrium has a full complement ofheat shock proteins. The menstrual cycle-dependent changes inthe amounts of heat shock protein suggest regulation by steroidhormones.     

Previously undetected Chlamydia trachomatis infection, immunity to heat shock proteins and tubal occlusion in women undergoing in-vitro fertilization.

The relationship between a previously undetected Chlamydia trachomatis infection, tubal infertility, immunity to heat shock proteins and subsequent in-vitro fertilization (IVF) outcome was evaluated. Women with tubal occlusion, with or without hydrosalpinges, and no history of C. trachomatis infection were tested for circulating antibodies to the human 60-kDa heat shock protein (Hhsp60), the C. trachomatis 10-kDa heat shock protein (Chsp10) and C. trachomatis surface antigens prior to their initial IVF cycle. Sera were obtained from 50 women whose male partners were infertile, 58 women with tubal occlusion but no hydrosalpinx and 39 women with tubal occlusions plus hydrosalpinx. Clinical pregnancies were documented in 68% of the women with male factor infertility. This was higher than the 43.1% rate in women with tubal occlusions (P = 0.04) and the 41% rate in women with hydrosalpinx (P = 0.02). C. trachomatis antibodies were present in one (2%) women with male factor infertility as opposed to 15 (25.9%) women with tubal occlusion (P = 0.003) and 13 (33%) with hydrosalpinx (P < 0.0001). Antibodies to Chsp10 were more prevalent in women with hydrosalpinx (46.8%) than in women with male factor infertility (P < 0.0001, 6%) or tubal occlusion (P = 0.0009, 15.5%). Hhsp60 antibodies were equally more prevalent in women with tubal occlusion plus (46.8%) or minus hydrosalpinx (41.4%) than in women with male factor infertility (P < 0.0002). Hhsp60 was more prevalent in those women positive for Chsp10 (P = 0.02) or C. trachomatis (P = 0.04) antibodies than in women lacking these antibodies. There was no relationship between any of the antibodies measured in sera and IVF outcome.     

Expression of heat shock protein 47 is increased in remnant kidney and correlates with disease progression

Glomerulosclerosis is characterized by accumulation of the mesangial extracellular matrix, including type I and IV collagen. The processing for the collagens in the glomeruli may play a critical role for development of glomerulosclerosis. We examined the expression of heat shock protein 47 (HSP47), a collagen-binding molecular chaperone in the progresive glomerulosclerosis model. Subtotally nephrectomized rats, unlike sham-operated rats, developed focal and segmental glomerulosclerosis. Immunological staining demonstrated an increased expression of HSP47 which paralleled the expression of type I and IV collagen in the glomeruli of the nephrectomized rats as the glomerulosclerosis developed. The mRNA levels encoding type I and type IV collagen and HSP47 were increased 3.4 fold, 3.6 fold and 2.8 fold, respectively, at week 7 after nephrectomy. By in situ hybridization, the expression of HSP47 mRNA was determined to be localized to the glomeruli with segmental sclerosis. These results suggest that HSP47 may play a central role in the process of extracellular matrix accumulation during the development of glomerulosclerosis.     

Salmonella typhimurium stimulation combined with tumour-derived heat shock proteins induces potent dendritic cell anti-tumour responses in a murine model

Appropriate activation of the immune system and effective targeting of tumour cells are the primary hurdles to be overcome for cancer immunotherapy to be successful and applicable to a wide range of tumour types. Our studies have examined the ability of bacterial-stimulated dendritic cells (DCs), loaded with tumour-associated antigens, to inhibit tumour growth in a murine model. Immature murine bone marrow-derived DCs were stimulated in vitro with the cytoplasmic fraction (CM) of Salmonella typhimurium in combination with heat shock proteins (hsps) from 4T1 tumours, isolated using heparin affinity chromatography. Activated DCs were administered subcutaneously. Tumours were generated by orthotopic inoculation of 4T1 cells in Balb/c mice. Primary tumour growth was measured using Vernier calipers, while lung metastases were measured using the clonogenic assay. S. typhimurium CM induced potent tumour necrosis factor (TNF)-alpha responses from DCs accompanied by significant up-regulation of CD80 and CD86 expression. When injected into mice, bacterial-stimulated DCs loaded with 4T1 hsps inhibited the formation of new 4T1 tumours and reduced the growth rate of established tumours. In addition, the number of lung metastatic nodules was reduced significantly in the DC-treated mice (1.6 +/- 0.6 versus 245.9 +/- 55.6, P = 0.0015). DCs stimulated with CM alone, exposed to tumour hsps alone or exposed to tumour hsps from an unrelated tumour cell line did not induce a protective immune response. Dendritic cells primed with a proinflammatory bacterial stimulus and tumour-associated antigens induce a protective anti-tumour immune response in this murine model.     

The involvement of heat shock proteins in murine liver regeneration

Partial hepatectomy （PHx） in mammals is a very common experimental model to investigate the process of liver regeneration. The surgery itself could give birth to a series of stresses, such as the temporary raise of body temperature and the ischaemia-reperfusion injury. Heat shock proteins （HSPs） were a family of stress-inducible proteins involved in maintaining cell homeostasis and regulating the immune system. In our study, we intended to investigate the expression and role of HSPs in liver regeneration. Using RT-PCR and Western blotting, we determined the expression in regenerating liver of HSP27, HSP60, HSP70 and HSP90 in mRNA level and protein level, respectively, with mice treated with sham operation as controls. We also used quercertin as an inhibitior of HSPs to explore their effects on liver regeneration. We found that hepatic expression of HSPs increased at the early phase of liver regeneration and declined to the constitutively low level later. Moreover, quercetin pretreatment delayed the progress of liver regeneration in mice via inhibition of HSPs. The results indicated that HSPs played an important role in liver regeneration. Cellular ＆ Molecular Immunology. 2007;4（1）：53-57.     

Successful primate immunization with peptides conjugated to purified protein derivative or mycobacterial heat shock proteins in the absence of adjuvants

We have previously shown in mice that antibodies can be induced to synthetic malaria peptides conjugated to mycobacterial antigens, such as purified protein derivative (PPD) or heat shock proteins (hsp), and given in the absence of adjuvants after a previous priming with bacille Calmette—Guérin (BCG). In the present study we investigated this model of immunization in the non-human primates, Saimiri sciureus monkeys. Monkeys primed with BCG subcutaneously and then immunized subcutaneously with the Plasmodium falciparum sporozoite (NANP)₄₀ synthetic peptide conjugated to PPD or mycobacterial hsp of 65 or 70 kD, in the absence of adjuvants, produced anti-peptide and anti-sporozoite IgG antibodies. Interestingly, the carrier effect of the hsp of 70 kD for the induction of anti-(NANP)₄₀ antibodies was also observed in the absence of a previous priming with BCG. These data suggest that such a vaccination strategy may be applied to humans.     

Heat shock proteins as carrier molecules: in vivo helper effect mediated by Escherichia coli GroEL and DnaK proteins requires cross-linking with antigen.

In the past few years we have shown that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a very strong helper effect in mice and monkeys when conjugated to peptides and oligosaccharides and given in the absence of adjuvants. In the present study we show that this adjuvant-free helper effect (i) is not due to lipopolysaccharide (LPS), since it was observed in LPS-resistant mice (C3H/HeJ) immunized with hsp-based constructs containing the malaria peptide (NANP)40, and (ii) is characteristic of hsp, since it was not observed with conjugates containing the mycobacterial p38 antigen, which is not a stress protein. Interestingly, the hsp GroEL and DnaK of Escherichia coli, which share a high degree of homology with the mycobacterial 65-kD and 70-kD hsp, respectively, exhibit a strong in vivo helper effect when conjugated to the (NANP)40 peptide, and the conjugates given in the absence of adjuvants. This in vivo helper behaviour of the GroEL and DnaK proteins corresponds well to that observed with the mycobacterial 65-kD and 70-kD hsp, respectively, since the hsp65- and GroEL-based constructs require previous priming of the animals with live bacille Calmette-Guérin (BCG), which is not needed for the hsp70- and DnaK-based constructs. Finally, using both mycobacterial and E. coli hsp we show that their in vivo helper effect in the absence of adjuvants requires cross-linking to the synthetic peptide. Taken together, our results suggest that the adjuvant-free helper effect observed with mycobacterial and E. coli hsp may be a generalized phenomenon, exhibited by hsp from diverse microorganisms. These findings may find applications in the design of vaccine constructs.     

Carcinosarcoma of the urinary bladder: Expression of epithelial markers and different expression of heat shock proteins between epithelial and sarcomatous elements

A case of carcinosarcoma composed of both adenocarcinoma and saarcomataus elements in the non-trigone region of the urinary bladder is presented. The epithelial element was a well to pooriy differentiated adenocarcinome with focal squamous metaplasia. The sarcomatous elements disclosed spindle cell sarcoma with focal epltheliold pattern and myxold change In the stroma, together with chondrosarcomatous and rhabdomyosarcomatous elements. By Immunohistochemical examination, not onty the carcinoma element but also the sarcomatous elements showed a positive lmmunoreaction for cytokeratin (CK), epithelial membrane antigen (EMA) and carcinoembryonic antigen. Some population of sarcomatous elements expressed smooth muscle actin and muscle specific actin (MSA) and a limited portion of epitheliold area showed a positive Immunoreaction for desmin, MSA and myoglobin, Indicating leiomyosarcomatous and rhabdomyosarcomatous differentiation, respectively. Unexpectedly, tumor cells In the chondrosarcomatws element revealed a simultaneous positvity of CK and EMA as well as S-100 protein. Both epithelial and sarcomatous elements showed an Intensive positive Immunoreaction for p53 and heat shock protein (HSP) 70. However, HSP27 and HSPGO were detected in most epitheilal elements and only in a small number of tumor cells in the sarcomatous area. These findings indicate that sarcomatous elements, Including heterologous elements, may derive from epithelial elements with partial or complete loss of epithelial features, and different factors other than p53 and HSP7O may associate wtth the morphological alteration of carcinoma.     

结核杆菌热休克蛋白70基因的克隆与原核表达

目的：克隆结核杆菌热休克蛋白70(TBhsp70)基因，并在大肠杆菌中表达。方法：利用PCR技术从结核杆菌H37Rv中扩增Hsp70基因，并将其克隆到pUC19中，进行测序。将得到的Hsp70基因克隆到表达载体pGEX-4T-1中，构建重组表达质粒pGEX—TBhsp70，在大肠杆菌DH5α中进行表达。结果：成功地克隆了TBhsp70基因。DNA测序证实，与GenBank公布的序列一致。含pGEX-TBhsp70基因表达质粒的大肠杆菌经IPTG诱导后，能够表达相对分子质量(MR)约为96000的融合蛋白。结论：获得了TBhsp70基因，成功地构建了原核表达质粒pGEX-TBhsp70，并在大肠杆菌得到表达，为其相关研究奠定了一定的基础。     

Lack of cytotoxic activity against Mycobacterium leprae 65-kD heat shock protein (hsp) in multibacillary leprosy patients.

Cytotoxic T cells play an important role in host defence mechanisms, as well as in the immunopathology of leprosy. In this study, we evaluated whether Mycobacterium leprae hsp18, hsp65 and Myco. tuberculosis hsp71 could induce cytotoxic T cell activity against autologous macrophages pulsed with these hsp. Paucibacillary (PB) patients and normal controls generated more effector cells than multibacillary (MB) patients with all three hsp tested. There was no cross-reactivity between any of the hsp tested. Mycobacterium leprae hsp65 induced cytotoxic responses only in those MB patients undergoing an erythema nodosum leprosum (ENL) episode. Although hsp65 and hsp18 induced similar proliferation in MB patients, a high proportion of these patients did not generate cytotoxic effector cells in response to hsp65. Hence, those T cells reacting to hsp65 may play an important role in the control of Myco. leprae infection.     

Andrology: Sperm hyaluronidase activation by purified predominant and major basic human seminal coagulum proteins

This study demonstrated that semenogelin I and II, the predominantand the major basic human seminal coagulum proteins respectivelywere shown to be potent activators of sperm hyaluronidase. Theseproteins stimulated the activity of bovine testicular hyaluronidase,goat cauda sperm hyaluronidase and human ejaculated sperm hyaluronidase.Human seminal plasma protein, which predominantly contains thebasic degradation residues of the basic coagulum proteins andalbumin (pI 4.7) and which is also basic at the assay pH 3.8,also showed activation of bovine testicular hyaluronidase whileacidic pepsin (pI < 1.0) exhibited no such activation. Thefindings may be utilized as an assay method for comparativeevaluation of specific activation units of the purified basicseminal coagulum proteins or their cleavage products and forquantifying the total basic protein (pI > 3.8) units in humanseminal plasma. One unit of the coagulum protein was definedas the amount of the activator that increases 1 mIU of hyaluronidaseactivity by 50%. The specific activity of the 57 kDa and 75–79kDa coagulum proteins, and that of serum albumin against Sigmabovine testicular hyaluronidase (type IV), were 11 447, 8600and 6252 units per mg protein respectively. It was speculatedthat human seminal coagulum proteins may have an activationeffect on spermatozoal hyaluronidase activity in vivo.