Presence of Antibodies to Heat Stress Proteins and its Possible Significance in Workers Exposed to High Temperature and Carbon Monoxide

Antibodies to the ubiquitous group of stress proteins known as heat shock proteins (Hsps) have been found to be associated with a number of diseases in humans. Hsps are known to be induced by certain xenobiotics, some of which are common in the working environment. The biological significance of the presence of such autoantibodies is presently unclear. In the present study, we used immunoblotting to investigate the presence of antibodies against the different stress proteins, Hsp27, Hsp60, Hsp71, Hse (heat shock cognate ) 73 and Hsp89a and D in groups of workers exposed to high temperature or carbon monoxide. These data were related to a detailed clinical evaluation and to various laboratory measurements including electrocardiogram (ECG), B echogram, white blood cell counts and typing, the activity of alanine aminotransferase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) and lymphocyte DNA damage. Antibodies to Hsp27 and Hsp71 were found more frequently in the high temperature and carbon monoxide-exposed groups than in controls (P (0.05 ). The carbon monoxide-exposed group showed the highest incidence of anti-Hsp antibodies. Anti-Hsp60 antibodies were only detected in workers exposed to high temperature or carbon monoxide. The percentage of workers with abnormal ECG, B echogram changes and displaying hepatitis B antigen (HBsAg ) was higher in the carbon monoxide group than in the control group (P＜0.05 ).There was a significant inerease in the activity of ALT in the high temperature and carbon monoxide groups and in the activities of ACP and ALP in the carbon monoxide group (P＜0.05 ). The extent of DNA damage measured in lymphoeytes was higher in workers from the high temperature and carbon monoxide-cxposed groups. We suggest that the increased frequeney of antibodies to Hsps is the result of these damages, of the release of denatured Hsps and of a decrease in the phagocytic ability of macrophages in these workers. The data gathered in the present study show a statistical relation between the occurrence of antibodies against Hsps and the frequency of health problems in workers and suggest a potential role for the antibodies as useful biomarkers to assess whether workers are experieneing environmental stress

Preliminary Studies on the Relationship betweenAutoantibodies to Heat Stress Proteins and Heat Injury of Pilots during Acute Heat Stress

Comparison in the heart rate, oral temperature and lymphocyte DNAdamage during heat stress was made in pilots with negative antibodies to heatstress proteins (HSPs) and those with positive antibodies in the man-made climate room with Western blot and comet assay. Our results showed that the increase in oral temperature, heart rate and lymphocyte DNA damage in pilots with the posi-tive antibodies to HSPs were higher than those in pilots with the negative antibod-ies during heat stress. These results indicated that the presence of autoantibodies in plasma of pilots might reflect heat damage and high sensitivity to heat.

Association of high expression in rat gastric mucosal heat shock protein 70 induced by moxibustion pretreatment with protection against stress injury

AIM:To study the effect of moxibustion on Zusanli or Liangmeng point on gastric mucosa injury in stress-induced ulcer rats and its correlation with the expression of heat shock protein 70 (HSP70). METHODS:Sixty healthy SD rats (30 males,30 females) were divided into control group,injury model group,Zushanli point group,Liangmeng point group. Stress gastric ulcer model was induced by binding cold stress method. Gastric mucosa ulcer injury (UI) index was calculated by Guth method. Gastric mucosa blood flow (GMBF) was recorded with a biological signal analyzer. Protein content and gene expression in gastric mucosal HSP70 were detected by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). Thiobarbital method was used to determine malondialdehyde (MDA) content. Gastric mucosal endothelin (ET) and prostaglandin E2 (PGE2) were analyzed by radioimmunoassay. RESULTS:High gastric mucosal UI index,high HSP70 expression,low GMBF and PGF2,elevated MDA and ET were observed in gastric mucosa of rats subjected to cold stress. Moxibustion on Zusanli or Liangmeng point decreased rat gastric mucosal UI index,MDA and ET. Conversely,the expression of HSP70,GMBF,and PGE2 was elevated in gastric mucosa after pretreatment with moxibustion on Zusanli or Liangmeng point. The observed parameters were significantly different between Zusanli and Liangmeng points. CONCLUSION:Pretreatment with moxibustion on Zusanli or Liangmeng point protects gastric mucosa against stress injury. This protection is associated with the higher expression of HSP70 mRNA and protein,leading to release of PGE2 and inhibition of MDA and ET,impairment of gastric mucosal index.

Expression of Heat Stress Protein 70 mRNA in Patients with Chronic Obstructive Pulmonary Disease and Its Significance

The effects of cigarette smoke extract (CSE) on the expression of heat stress protein 70 (Hsp70) in human bronchi smooth muscle cells were investigated in vitro, and the changes in Hsp70 mRNA in the patients with chronic obstructive pulmonary disease and their significance were explored. Human bronchi smooth muscle cells were cultured with CSE at the different concentrations. The expression of Hsp70 mRNA and Hsp70 was detected by reverse translation-polymerase chain reaction (RT-PCR) and Western blotting respectively. Levels of Hsp70 mRNA and Hsp70 in lymphocytes from 20 patients with COPD and 20 healthy smoking control subjects were measured by RT-PCR and Western blotting. The results showed the expression of both Hsp70 mRNA and Hsp70 was decreased conformably in human bronchi smooth muscle cells treated with CSE at certain concentration in vitro. The A values of the Hsp70 mRNA expression were 0.24±0.11 and 0.42±0.13 respectively in COPD patients and healthy smoking controls with the difference being significant (P<0.01). There was also significant difference in the A values of the Hsp70 expression between COPD patients and healthy smoking controls (20.9±9.9 vs 44.8±15.3, P<0.01). The levels of Hsp70 mRNA had strongly positive correlation with Hsp70 protein (r = 0.85, P<0 01). It was suggested that the expression of Hsp70 mRNA was in concordance with the expression of Hsp70, which could provide a basis on the study of Hsp70 gene regulation and Hsp70 gene in the development of COPD.

春尺蠖热激蛋白AcinHsp70基因的克隆、分子特性与表达分析

【目的】克隆春尺蠖热激蛋白Hsp70基因,分析其理化性质与表达情况,探究该基因在春尺蠖应对外界不利环境中的作用。【方法】基于前期所测转录组数据(春尺蠖幼虫不同低温胁迫),克隆获取Hsp70基因,命名为AcinHsp70(GenBank登录号:OP750249);利用生物信息分析软件对Hsp70基因的理化性质进行分析;采用qPCR技术检测AcinHsp70基因在不同温度(-10、-5、0、5和25℃)胁迫中回温与不回温及4℃不同时间处理下的表达谱。【结果】春尺蠖AcinHsp70基因完整CDS序列全长1899 bp,编码633个氨基酸,蛋白质预测分子量为69.91 kDa,预测等电点为5.51。在N端均不含信号肽且无跨膜区。磷酸位点检测分别发现丝氨酸、苏氨酸和酪氨酸磷酸位点24、26和8个;未发现糖基化位点。该蛋白的平均亲水性为-0.507,预测不稳定系数为33.19,故该蛋白属于亲水稳定蛋白,亚细胞定位结果显示,其主要位于细胞质。序列一致性比对与系统进化树分析结果显示,春尺蠖与庆网蛱蝶(Melitaea cinxia)亲缘关系最近,序列一致性达92.91%。基因表达结果显示,不同温度(-10℃除外)胁迫下,AcinHsp70基因表达量均无明显差异,回温后表达量显著上调。AcinHsp70基因表达量总体呈先升后降趋势,在4℃处理1 h后表达量达到最大值。【结论】AcinHsp70基因与春尺蠖应对低温胁迫的响应密切相关,结果有助于揭示该基因在低温胁迫下的分子功能。

Hsp70,Toll样受体4抗体与自身免疫性内耳病的关系

目的:建立一种自身免疫性内耳病的动物模型,寻求Toll样受体4抗体与Hsp70的关系.方法:提取豚鼠内耳膜迷路组织为抗原.将豚鼠用环磷酰胺预处理,将抗原与等量弗氏完全佐剂一起免疫豚鼠,观测Hsp70在内耳的表达;通过鼓室注射Toll样受体4抗体,再观测Hsp70在内耳的表达.结果:免疫豚鼠后听性脑干反应阈显著提高,内耳出现显著的炎细胞浸润,Hsp70的表达增强,TNF-α,NF-κB的表达增多,血清中检测到抗Hsp70抗体;加入Toll样受体4抗体后Hsp70的表达减弱,炎细胞减少,TNF-α,NF-κB的表达降低.结论:Toll样受体4抗体可下调Hsp70在自身免疫性内耳病中的表达.

血清热休克蛋白70、热休克蛋白90和胃泌素水平对重症颅脑损伤并发应激性溃疡的预测

目的探讨血清热休克蛋白70(HSP70)、热休克蛋白90(HSP90)和胃泌素(GAS)水平对重症颅脑损伤并发应激性溃疡的预测价值。方法选取126例重症颅脑损伤患者,入院后均采用酶联免疫法(ELISA)检测血清HSP70、HSP90和GAS水平,并实施标准去大骨瓣减压术联合腰大池引流术。统计应激性溃疡发生率,分析血清HSP70、HSP90和GAS水平与患者并发应激性溃疡的关系及三者联合对该并发症的预测价值。结果应激性溃疡发生率为32.17%;并发应激性溃疡患者入院后血清HSP70、HSP90和GAS水平均高于未并发者(P<0.05);年龄、入院格拉斯哥昏迷量表(GCS)3~5分、入院后血清HSP70水平、入院后血清HSP90水平、入院后血清GAS水平、入院后转铁蛋白(TRF)水平、入院后随机血糖水平、低血压、低氧血症、代谢性酸中毒、应用糖皮质激素均是并发应激性溃疡的危险因素(P<0.05),入院后血红蛋白(Hb)水平、入院后红细胞压积(HCT)、应用保护胃黏膜药物、应用乌司他丁是其保护因素(P<0.05);血清HSP70、HSP90、GAS水平联合预测重症颅脑损伤并应激性溃疡的灵敏度均高于单独预测(P<0.01),曲线下面积(AUC)均高于单独预测(P<0.05),特异度均与单独预测差异无统计学意义(P>0.05)。结论血清HSP70、HSP90、GAS水平升高可增加重症颅脑损伤并发应激性溃疡的风险,且年龄、入院GCS3~5分等也均是其危险因素,入院后Hb水平、入院后HCT等是其保护因素,血清HSP70、HSP90、GAS水平联合预测并发应激性溃疡的效能高。

Xiaotan Tongfu granules contribute to the prevention of stress ulcers

AIM:To investigate the efficacy and potential mechanism of Xiaotan Tongfu granules(XTTF)in stress ulcers.METHODS:One hundred sixty rats were randomly divided into 4 groups(n=10)as follows:the model group(MP group),the control group(CP group),the ranitidine group(RP group)and the XTTF granule group(XP group).Rats in the MP group received no drugs,rats in the CP group received 0.2 mL of a 0.9%sodium chloride solution via oral gavage,and rats in the RP and XP groups received the same volume of ranitidine(50 mg/kg)or XTTF granule(4.9 g/kg).The cold-restraint stress model was applied to induce stress ulcers after 7 consecutive days of drug administration.Afterwards,rats were sacrificed at 0,3,6 and24 h.Gastric pH was measured by a precise pH meter;gastric emptying rate(GER)was measured by using a methylcellulose test meal;myeloperoxidase activity(MPO),macrophage migration inhibitory factor(MIF),proliferating cell nuclear antigen(PCNA),and heat shock protein 70(HSP70)were measured by immunohistochemical staining;and mucosal cell apoptosis was measured by transferase dUTP nick end labeling.RESULTS:In the cold-restraint stress model,the development of stress ulcers peaked at 3 h and basically regressed after 24 h.Gastric lesions were significantly different in the RP and XP groups at each time point.Interestingly,although this index was much lower in the RP group than in the XP group immediately following stress induction(7.00±1.10 vs 10.00±1.79,P<0.05.Concerning gastric pH,between the RP and XP groups,we detected a statistically significant difference immediately after stress induction(0 h:4.56±0.47 vs 3.34±0.28,P<0.05)but not at any of the subsequent time points.For GER,compared to the RP group,GER was remarkably elevated in the XP group because a statistically significant difference was detected(3 h:46.84±2.70 vs 61.16±5.12,P<0.05;6 h:60.96±6.71 vs 73.41±6.16,P<0.05;24 h:77.47±3.17 vs 91.31±4.34,P<0.05).With respect to MPO and MIF,comparisons between the RP and XP groups revealed statistically significant differences at 3 h(MPO:18.94±1.20 vs 13.51±0.89,P<0.05;MIF:150.67±9.85 vs 122.17±5.67,P<0.05)and 6 h(MPO:13.22±1.54 vs 8.83±0.65,P<0.05;MIF:135.50±9.46 vs 109.83±6.40,P<0.05).With regard to HSP70,HSP70 expression was significantly increased in the RP and XP groups at 3 and 6 h compared to the MP and CP groups.In addition,comparing the RP and XP groups also showed statistically significant differences at 3 and 6 h.The expression of PCNA was higher in the RP and XP groups 3 h after stress induction.Between these two groups,small but statistically significant differences were observed at all of the time points(3 h:69.50±21.52 vs 79.33±15.68,P<0.05;6 h:107.83±4.40 vs 121.33±5.71,P<0.05;24 h:125.33±5.65 vs 128.50±14.49,P<0.05)except 0 h.With regard to apoptosis,the apoptotic activity in the RP and XP groups was significantly different from that in the MP and CP groups.The XP group exhibited a higher inhibition of cell apoptosis than the RP group at3 h(232.58±24.51 vs 174.46±10.35,P<0.05)and6 h(164.74±18.31 vs 117.71±12.08,P<0.05).CONCLUSION:The Xiaotan Tongfu granule was demonstrated to be similar to ranitidine in preventing stress ulcers.It exhibited multiple underlying mechanisms and deserves further study.

The Combined Effects of High Temperature and Carbon Monoxide on Heat Stress Response

In this study,we have examined the effects of exposure to high temperature, carbon inonoxideor a combination of both conditions in a model system,the rat and in industrial workers.In the rat liver, HSP70 mRNA and HSP70 synthesis were measured by dot hybridization and western blot. The results showed that after a heat stress HSP70 mRNA and its product, HSP70 increased significantly and there was a synergism in the combined effects of high temperature and carbon monoxide exposure on the induction of HSP70 mRNA and HSP70 synthesis. Heat played a major role in this induction. The presence of antibodies to human HSP27, HSP60, HSP70,HSC73, HSP89 αand β in workers exposed to heat, carbon monoxide was also measured by western blot using purified HSPs as antigens. Plasma free amino acids were measured in the saine group of workers. The incidence of antibodies to HSP27 and HSP70 Was significantly higher in the workers working in an environment with extreme heat, and high carbon monoxide ernission than in a control group. The carbon monoxide exposed group showed the highest incidence of antibodies to HSPs. Although our previous results indicated that workers had an insufficient protein intake,plasma free amino acids tended to increase, especially in methionine and tryptophan two kinds of amino acids which are absent from the main stress protein, HSP70.These results suggest that the major problems that these workers may face are how to facilitate the use of plasma free amino acids and reduce the inhibition of synthesis of normal proteins when they are exposed to occupational harmful factors.These resultsalso add new information on the measurement of HSPs as a potential biomonitor to assess whether organisms are experiencing metabolic stress within their environment.

Neuroprotective effect of heat shock protein 70 Inhibition of Tau protein expression

BACKGROUND： Previous studies have shown that heat shock protein 70 （HSP70） has neuroprotective effects by decreasing phosphorylation of Tau protein, thereby reducing the expression of Tau protein and proper aggregation. OBJECTIVE： To observe and verify expressional changes of HSP70 and Tau in retinal ganglion cells following stretch injury to the right optic nerve in rats, and to determine the effect of heat stress pretreatment on HSP70 and Tau protein expressions. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Neurology Research Institute of the First Affiliated Hospital of Chongqing Medical University from March to June 2006. MATERIALS： Instant SABC immunohistochemistry kit, as well as mouse anti-HSP70 and rabbit anti-Tau polyclonal antibodies, were purchased from Wuhan Boster Bioengineering Limited, China. METHODS： A total of 57 male, Wistar rats were randomly assigned to 4 groups： control （n = 3）; 150-180 g stretch force was induced in the right optic nerves in stretch-only group （n = 18） to establish optic nerve stretch injury model; heat stress was applied to 18 animals in heat-stress treatment group; 18 rats in the heat-stress pretreatment plus stretch group were subjected to identical stretch injury as stretch-only group after 24-hour heat-stress pretreatment. According to sacrifice time, the groups were assigned to 6 subgroups at different time points of 4, 8, and 16 hours, and 1,3, and 5 days, with 3 rats in each subgroup. No treatment was performed in the control group except anesthesia. MAIN OUTCOME MEASURES： Morphological changes of optic nerves and retinal ganglion cells following stretch injury were observed by light microscopy following hematoxylin-eosin staining. HSP70 and Tau protein expression levels were observed in retinal ganglial cells from each group using im munohistochemistry. RESULTS： （1） Compared with the control group, morphological axonal and retinal ganglial cell changes, as well as a decreased number of retinal ganglial cells, were identified in the stretch-only group （P 〈 0.01）. Pathological damage in optical nerve and retinal ganglial cells were not remarkable in the heat-stress pretreatment plus stretch group, with no statistical difference in the number of retinal ganglial cells compared with the control group （P 〉 0.05）. （2） Compared with the control group significantly increased HSP70 expression in retinal ganglial cells occurred in the stretch-only, heat-stress treatment, and heat-stress pretreatment plus stretch groups （P 〈 0.05 or P 〈 0.01 ）. The peak of HSP70 expression was earlier in the heat-stress pretreatment plus stretch group compared with the stretch-only and heat-stress treatment groups, and was expressed over a longer period of time compared with the heat-stress treatment group. Compared with the control group, Tau expression in the retinal ganglial cells rapidly increased 4-16 hours following stretch injury in the stretch-only group （P 〈 0.05 or P 〈 0.01）, and obviously decreased in the heat-stress pretreatment plus stretch group （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Tau expression increased following stretch injury, with an earlier expression peak than HSP70, which indicated that stretch injury-induced HSP70 expression was not strong or quick enough to sufficiently protect the nerve. A much more enhanced HSP70 expression, with an earlier peak and longer expression period, was observed in rats subjected to stretch injury following heat stress, which demonstrated that HSP70 exhibited neuroprotective functions by reducing abnormal aggregation of Tau.

热休克蛋白70促进脊髓损伤恢复研究进展

脊髓损伤(SCI)是由椎骨骨折或脱位造成的一种高致残率疾病,严重降低患者生活质量。热休克蛋白(Hsps)是正常的细胞内蛋白,在细胞受到应激或损伤时大量产生。热休克蛋白70(Hsp70)是热休克蛋白家族中的重要成员,该蛋白已被证明在SCI后的继发性损伤调节中起着关键作用。故对SCI后,Hsp70的产生和作用机制进行综述,以期为后续相关研究提供参考。

复方玄驹胶囊联合二甲双胍、氯米芬治疗多囊卵巢综合征的效果及对氧化应激指标、HSP70、sFlt-1水平的影响

目的分析复方玄驹胶囊联合二甲双胍、氯米芬治疗多囊卵巢综合征的效果。方法按照电脑盲选法将我院诊治的86例多囊卵巢综合征患者分为对照组(43例,二甲双胍+氯米芬)与研究组(43例,复方玄驹胶囊+二甲双胍+氯米芬)。比较两组的治疗效果。结果治疗3个月后,研究组的总抗氧化能力(TAC)、谷胱甘肽过氧化物酶(GPx)、超氧化物歧化酶(SOD)、可溶性血管内皮生长因子受体1(sFlt-1)、性激素结合球蛋白(SHBG)水平高于对照组,热休克蛋白70(HSP70)、血管内皮生长因子(VEGF)、血管紧张素Ⅱ(AngⅡ)、肝细胞生长因子(HGF)、硫酸脱氢表雄酮(DHEA-S)、抗缪勒管激素(AMH)水平低于对照组(P<0.05)。两组的不良反应总发生率无显著差异(P>0.05)。结论复方玄驹胶囊联合二甲双胍、氯米芬治疗多囊卵巢综合征的效果显著,能够改善患者的氧化应激反应、血管活性因子水平,调节HSP70、sFlt-1水平,临床可进一步推广应用。

大黄蒽醌提取物对罗氏沼虾高温下抗氧化能力与热应激蛋白70基因表达的影响

将罗氏沼虾随机分成5组,每组3个平行,每个平行1000尾,第1组为对照组,投喂基础日粮;另外4组为试验组,在基础日粮中分别添加0.05%、0.1%、0.2%、0.4%大黄蒽醌提取物。饲养10周后,对罗氏沼虾进行连续35℃高温应激48h,测定肝胰腺总抗氧化能力、过氧化氢酶、超氧化物歧化酶、丙二醛、一氧化氮以及应激蛋白70的mRNA相对含量变化。结果表明:应激前,0.05%组显著提高了肝胰腺过氧化氢酶活性、超氧化物歧化酶活性,显著降低了肝胰腺丙二醛含量;0.2%试验组显著增加了肝胰腺总抗氧化能力、一氧化氮浓度,显著降低了肝胰腺丙二醛含量;0.10%、0.2%试验组显著增加HSP70mRNA的相对含量;高温应激后,各组肝胰腺总抗氧化能力、过氧化氢酶活性、超氧化物歧化酶活性、一氧化氮浓度呈现降低趋势,其中0.1%和0.2%大黄蒽醌提取物相对较高,对照组较低;肝胰腺丙二醛含量呈现增加趋势,其中加0.1%和0.2%大黄蒽醌提取物比对照组低。应激6h后0.1%和0.2%大黄蒽醌提取物组的HSP70mRNA的相对含量仍比对照组高。因此添加0.1%和0.2%大黄蒽醌提取物提高了虾的抗氧能力,促进HSP70mRNA的相对含量,对高温应激有一定的保护作用。

苯并[a]芘对内皮细胞增殖活性的影响及与热应激蛋白70表达关系的研究

为探讨苯并 [a]芘 (BaP)对猪主动脉内皮细胞增殖活性的影响以及与热应激蛋白 70 (HSP70 )表达的关系。采用离体培养猪主动脉内皮细胞 ,不同浓度BaP(0、0 5、1、10、2 0 μmol L)染毒 2 4h ,以MTT法分析不同浓度BaP对内皮细胞增殖活性的影响并以Western blot法检测内皮细胞HSP70表达的改变。结果发现 ,BaP染毒 2 4h后 ,主动脉内皮细胞增殖活性随染毒剂量的增加而逐渐降低 ;低剂量 (0 5 μmol L)染毒组HSP70的表达与对照组相比无明显差异 ,而中、高剂量 (1、10、2 0 μmol L)抑制HSP70的表达。结果提示BaP抑制主动脉内皮细胞增殖生长活性 ;一定浓度的BaP能抑制主动脉内皮细胞HSP70的表达。

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。

急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70表达的影响

目的:探讨急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70(heat shock protein 70,HSP70)表达的影响。方法:将32只8周龄雄性小白鼠随机均分为4组,饲养7d后,进行热应激处理,温度控制在(39±0.5)℃,时间分别为0.5、1和3h。应激后立即采血,分离血清测定谷草转氨酶(GOT)含量。一侧附睾制备精子悬液,用于计算精子密度和顶体畸形率;另一侧附睾、睾丸、输精管用于免疫组化研究。结果:应激后,小鼠体重、睾丸系数、顶体畸形率变化不显著(P>0.05),附睾系数和精子密度有不同程度的下降,GOT含量急剧升高(P<0.01)。随着应激时间的延长,小鼠精子密度呈递减趋势,顶体畸形率呈上升趋势。应激时间最短的0.5h组小鼠体重、睾丸系数、附睾系数的降幅反而最大。免疫组化法观察发现,HSP70在性成熟小鼠睾丸、附睾、输精管中均有表达。正常状态下,HSP70在睾丸组织间质细胞中少量表达,应激后分布于间质细胞核,此外在精母细胞核与精子细胞核中也有大量分布;附睾中HSP70主要分布于主细胞质,基细胞和亮细胞中没有表达,应激后附睾体的纤毛细胞中也发现大量棕色颗粒;输精管中HSP70主要定位在基细胞质,主细胞中不表达。随着应激时间的延长,HSP70在睾丸、附睾中的表达量明显升高,而在输精管中的增幅不明显。结论:急性热应激对性成熟雄性小鼠的生殖系统造成了损伤;HSP70在睾丸、附睾、输精管中的表达与定位具有区域特异性和细胞特异性,提示其可能参与精子的发生与成熟;HSP70在应激状态下表达量大幅上升的作用可能在于保护细胞免受高热损伤。

猪肺炎霉形体热休克蛋白Hsp70单克隆抗体的制备及鉴定

以肺炎霉体体(Mhp)Yin-1株为模板,利用所合成的引物扩增了伴侣蛋白DnaK(热休克蛋白Hsp70)C末端基因,将得到的目的片段分别克隆至表达载体pET-28a和pGEX-6P-1中,原核表达获得了重组蛋白6P-1-DnakC和28a-DnakC,经Western-blot分析,均能被Mhp抗血清识别。以28a-DnakC作为包被抗原,建立了间接ELISA检测方法,以6P-1-DnakC为免疫抗原,免疫BALB/c小鼠。筛选和鉴定了2株分泌抗DnaK的杂交瘤细胞株,命名为1AD10、2AF11。Western-blotting结果表明,这2株单抗与Mhp发生特异性反应而不与其他相关霉形体发生反应。抗体亚类鉴定结果显示,1AD10、2AF11分别为IgG2b和IgG2a亚类,且轻链均为κ链。相加ELISA试验表明,2株单抗能识别不同的抗原表位。

热休克蛋白70对氧化应激所致核仁素裂解的影响

为探讨热休克蛋白 70对氧化应激 (0 .5mmol L过氧化氢 )所致核仁素裂解的影响及其机制 ,采用免疫印迹技术检测氧化应激诱导核仁素的裂解 ;通过热休克反应和构建热休克蛋白 70转基因细胞观察热休克反应与热休克蛋白 70对核仁素裂解的影响 ,同时采用免疫共沉淀技术检测热休克蛋白 70与核仁素之间的相互作用。结果发现 ,0 .5mmol L过氧化氢可导致多种细胞的核仁素发生裂解 (有 80kDa裂解片断出现 ) ,而且最早出现裂解片段的时间都在过氧化氢处理 30min到 1h左右 ;热休克预处理及转热休克蛋白 70后均引起热休克蛋白 70的表达明显增加 ,同时显著抑制抑制氧化应激所致核仁素裂解片段的出现 ,免疫共沉淀结果显示在氧化应激时热休克蛋白70与核仁素直接结合 ,形成复合物。以上结果提示 ,热休克反应与热休克蛋白 70可以显著抑制氧化应激所致核仁素的裂解 ,其机理与氧化应激时热休克蛋白 70与核仁素直接结合有关。

热应激对肉鸡的影响及热休克蛋白70的研究进展

在现代化畜禽生产中,热应激已成为制约肉鸡生长、代谢、肉品质及生理功能的重要因素之一,给养殖业带来了严重的经济损失。热应激蛋白(heat shock protein,HSPs)是动物在不良因素作用下所产生的一组特异性蛋白质,而HSP70是其家族成员中最保守、最重要的蛋白之一;它具有分子伴侣、热耐性,抗过氧化损伤等特性,能够使机体迅速适应环境变化,在热应激保护中发挥了重要的作用。作者主要阐述了热应激对肉鸡的影响和HSP70的生物学特点及其抗热应激的作用机理,以期为从药物方面探讨HSP70的抗应激功能提供理论依据。

慢性应激抑郁模型大鼠强迫游泳后海马中Hsp70的表达

目的研究慢性应激对强迫游泳大鼠海马神经元热休克蛋白70(Hsp70)表达的影响。方法将50 只大鼠随机分为实验组和对照组各25只。实验组大鼠通过21天的应激刺激制作抑郁动物模型,此期间对照组大 鼠正常饲养。此后所有大鼠逐只进行急性强迫应激刺激。采用特异性抗体的免疫组织化学方法,观察2组大鼠在 强迫游泳后2 h、6 h、18 h、24 h和48 h各时点海马神经元Hsp70的表达情况。结果慢性应激抑郁大鼠模型接受 急性强迫游泳应激后,海马CA3区和齿状回(DG)内Hsp70蛋白的表达较对照组强迫游泳后显著降低(P<0．05)。 结论慢性应激使大鼠在急性强迫游泳应激后海马CA3区和DG内Hsp70的表达降低。