【作者】 吴晓岚；

【导师】 赵书芬；

【作者基本信息】 中国医科大学 ， 生理学， 2005， 硕士

【摘要】 前言 将机体置于热环境时可致体温升高。近年来的研究证明,机体或组织细胞暴露于热环境时可诱导产生热休克蛋白70(HSP70),HSP70是一种非特异性细胞保护蛋白,具有分子伴侣功能,能够增强细胞对热损伤的抵抗能力,并修复热损伤变性的蛋白质。 一般情况下,当体温达到一定的高度(41℃)时,就不会再随着内外环境刺激而继续升高,这种现象称为热限。提示体内存在调控体温的自限机构,但其机制尚不十分清楚。AVP是一种内生性解热物质,在脑内最敏感的作用部位是腹侧中隔区(VSA)。IL-1β作为内生致热原可致体温升高,有文献报告向VSA注射AVP能显著地抑制家兔的IL-1β性发热反应。本研究旨在观察热应激后下丘脑内大量表达的HSP70对IL-1β性发热反应以及对下丘脑和VSA中AVP含量的影响。以探讨HSP70是否参与热限作用。 材料与方法 1.动物和手术 健康雄性S-D大鼠,体重200-250g,基础体温(37.5±1.0)℃。室温(22±2)℃,湿度(50±2)%,光照12h:12h,单笼饲养,自由进食水。在盐酸氯胺酮和盐酸甲苯噻嗪的联合麻醉下,参照大鼠脑图谱,用立体定位仪向一侧下丘脑视前区(POA)插入一规格为23号不锈钢管,并留置与之相匹配的针芯,用牙科水泥固定。术后大鼠单笼饲养,常规喂养一周后进行实验。实验前对大鼠进行测温服习7d。测量体温时将温度探头插入大鼠直肠6cm处,待数值稳定后读数,以给药前1小时测得的三次直肠温的平均值为大鼠基础体温。实验过程中记录大鼠体温(0-1h内每隔15min,1-2h内每隔30min,2-8h内每隔60min测量一次)。实验结束后检查插管位置,舍更多还原

【Abstract】 INTRODUCTIONBody temperature is increased when the body is in hot environment. Recent studies demonstrate that the expression of heat shock protein 70(HSP70) is induced by exposure of cells and tissues to thermal conditions. HSP70 is a kind of non - specific cytoprotective protein, which has been shown to function as molecular chaperone, and can enhance cells to protect against heat - induced damage and repair heat - damaged and/or denatured proteins.Generally, body temperature doesnt go on increasing with the stimula of internal and external environment when it arrives at certain height,which is characterized as febrile ceiling. It indicates that there is a self - limitation structure for thermoregulation in the body, but the mechanism is still unclear. AVP is an endogenous antipyretic and the most sensitive active site is ventral septal area (VSA) in brain region. IL - 1β can elevate body temperature as endogenous pyrogen. It was reported that IL - 1β - induced fever response can be remarka-blely inhibited by the injection of AVP into VSA in rabbit. The present study aims to observe the effect of the expression of HSP70 in hypothalamus on IL - 1β - induced fever response and AVP content in hypothalamus and VSA after heat stress, so as to explore whether HSP70 takes part in the role of febrile ceiling.MATERIALS AND METHODS1. Animals and surgeryHealthy male Sprague - Dawley rats, weighing 200 - 250g, with basic body temperature(37. 5 ±1.0) ℃ were served for all the experiments. During theseexperiments,rats were individually housed in plastic cages at (22 ±2)℃. ambient temperature and (50 ± 2) % humidity and maintained on a 12:12 - h light -dark cycle with lights. Food and water were available ad libitum. Under intraperitoneal anesthesia ( ketamine hydrochloride and acepromazine maleate) , a 23 size of stainless steel cannula was implanted into the preoptic - anterior hypothalamus area using stereotaxic apparatus according to the brain atlas of rat, retained needle which was matched with it,and then anchored with dental acrylic. After operation, animals were returned to their home cages for recovery with free access to food and water for 7 days. Rats were habituates to experimental proce-dures by noninvasive handling daily for 7 consecutive days before the experiments. Digital thermometer probe was inserted 6 centimeters in ratsrectum when temperature was measured, and then the value was recorded after stabilization. Mean of three values 1 hour before administration was taken as baseline of pre -handling. After administration, rectal temperature was recorded respectively during experiments(to measure once every other 15 min in 0 - 1h, every other 30 min in 1 -2h, every other 60 min in 2 -8h). When the experiment was over, cannula’s positions were checked to discard incorrect ones.2. Expression of HSP70 assay in hypothalamus tissue of rats Hypothalamus tissue was taken, homogenated and centrifuged. Then the su-pernatants were used to measure the expression of HSP70 by Western blotting.3. AVP levels assay in brainRats were put to death immediately at the peak of IL - 1 β - induced fever , and the brain were taken to boil 5 minutes in boiling physiological brine . Hypothalamus and VSA tissues 30mg were respectively weighted, homogenated , centrifuged and assayed concentration of AYP with radioimmunoassay.4. Statistical analysisResults were presented as mean ± SD . Change in body temperature were presented and analyzed with △T (℃) and thermo -response index . Data were analyzed using T - test.RESULTS1. HSP70 expression was induced increasingly after heat stress and reached peak at 12 hours;if rats were injected cycloheximide(CHX) before heat stress, HSP70 expression in hypothalamus was obviously inhibited at each time.2. Fever response was induced by the injection of IL - 1β into POA in rats, at the same time, AVP content in VSA was significantly increased.3. When rats were administrated IL -1β after heat stress, the extend of IL -1β - induced fever was obviously reduced, and AVP content in VSA wasn t significantly increased.4. When rats were injected CHX before heat stress,the extend of IL - 1β -induced fever wasnt obviously reduced,and AVP content in VSA was significantly increased.DISSCUSSIONThe experiment used IL-1β to make the febrile animal model of rat. Body temperature began to rise 15 minutes after administration in rats. This quick elevation of body temperature could have relation with direct administration into POA, and the peak of fever was at 2 hours after administration. AVP was an endogenous antipyretic, which was synthesized by hypothalamus, and conveyed to VSA through nerve fibres between hypothalamus and septal area to develop thermoregulation function. The experiment detected that AVP content in VSA was significantly higher in IL-1β and CHX + HS + IL - 1β groups than control group in the peak time of fever; However, AVP content in hypothalamus was slightly increased, but wasnt significantly higher than control group. This was consistent with previous report.It was reported that heat stress could induce HSP70 expression,which could enhance thermotolerance of cells. In this experiment, HSP70 expression was induced increasingly by heat stress, and then endogenous pyrogen IL-1β was administrated . It was obvious that the curve of body tempetature was low - flat,更多还原