【作者】 马中女；

【导师】 曹宇；

【作者基本信息】 中国医科大学 ， 生理学， 2004， 硕士

【摘要】 目的 热休克转录因子，也被称为热休克因子(heat shock factor，HSF)，是一组结构和功能具有广泛同源性，普遍存在于真核生物细胞中的蛋白质。其中HSF1被称为快速的应激反应因子，对各种应激源刺激均敏感，由存在于胞浆中的无活性的单体形态向三聚体转化，并转入细胞核，使丝氨酸残基磷酸化，具备与hsp70基因上游的热休克元件(heat shock element，HSE)结合的能力，调控hsp70基因的表达，促使hsp70基因的转录和翻译。 研究发现，许多传染源或致炎刺激物侵入体内后激活产生内生致热原细胞诱生内生致热原(endogeny pyrogen，EP)，进而引起发热。EP主要由单核巨噬细胞群产生，包括IL-1、TNF等，EP的释放是发热的关键环节。 通过对hsp70、IL-1β和TNF-α基因启动子同源性调查发现，IL-1β基因的启动子中也含有与hsp70基因上游相同的完整的HSE序列，TNF-α基因中临近启动子85个核苷酸序列和5端非翻译区中含有多个不完整的HSE序列。因此，可以推测活化的HSF1不仅能与hsp70基因上游的HSE结合，而且也可能与IL-1β、TNF-α基因上游的HSE或HSE相似的序列结合，从而影响IL-1β、TNF-α基因的表达。那么机体发热时HSF是否通过影响内生致热原基因IL-1β、TNF-α等的转录而参与调控体温呢?有关此方面的研究迄今未见报道。 为探讨HSF1在体温调节中的可能作用，本研究着眼于在发热过程中HSF1的表达对IL-1β、TNF-α基因转录水平的影响，应用脂多糖(LPS)复制家兔发热模型，观察发热的不同时相血单核细胞中HSF1的表达和IL-1β、TNF-αmRNA表达以及二者之间变化的时相关系。本研究对进一步寻找机体抑热机构，明确热限机制具有重要的意义。 实验方法 1．动物分组、模型及标本制备、项目检测： 家兔随机分为6组，第I组是对‘照组，静脉注射与实验组US同体积的生理盐水后160niln时取材，第n、111、W、V、VI是实验组，静脉注射IJS后分别在80、160、240、320、400而n时取材;经家兔耳正中动脉取血10血，以Histop叔IUe密度剃度离心获取单个细胞，在利用单核细胞的豁附特性进一步分离获得单核细胞;用RT一PCR半定量方法检测IJS发热不同时相单核细胞IL一lp和翎F一amRNA表达量，以W比tem blot分析IJS发热不同时相单核细胞HSFI的表达量。 2.图象处理: PCR结果拍成黑白照片，用电泳凝胶成像分析仪(Che面Im卿的5加)扫描，用其中的凡uor Chen v2.0分析软件分析各DNA扩增片段的密度功V值，将每一种目的DNA扩增片段密度与相应的内参照G赶勺H扩增片段密度的比值(e/C)作为其mRNA水平的定量指标;同样也用电泳凝胶成像分析仪(Che而Imager5500)扫描硝酸纤维素膜，用其中的nuOIChen v2.0分析软件测定各组蛋白质带的IDV值，作为ESFI的相对含量。 3.统计分析; 所用实验数据均用均数土标准差(又土s)表示，各组均数间显著性差异的比较用t检验，相关分析用相关性检验分析法，应用SAS统计软件。实验结果 1.注射L咫后家兔的体温变化: 给家兔静脉注射L玲(0.5林岁kg)可引起双峰热，注射IJS后印min时动物体温上升达第一个高峰1 .3土0.32℃，之后略有回降，在注射L玲后150而n时动物体温上升达第二个高峰1 .98士0.36℃，36Omin之内可回降至正常水平。发热组与对照组比较差异十分显著(p<0.01)。 2.1‘玲发热不同时相，单核细胞中IL一lp和TNF一仪mRNA含量的变化: 单核细胞中几一1 pmRNA含量在注射us后so而n开始升高，1印而n时达高峰，400min时回降至基础水平，与对照组相比差异显著(p<0.01);单核细胞中TNF一amRNA含量在注射IJS后son‘n时达高峰，然后逐渐下降，320而n时回降至基础水平，与对照组相比差异显著(p<0.01)。 3.L玲发热不同时相，单核细胞中HSFI表达量:单核细胞中HSFI的总含量(包括三聚体和单体)在实验观察期间无明显变化(p>0.05);而HSFI三聚体含量随体温的升高逐渐增加，直到体温降至正常水平，与对照组相比差异显著(p<0.01)。 4.1尸S发热时单核细胞中达量的关系:HSFI表达量与IL一lp和TNF一仪mRNA表相关分析表明:I‘巧发热时(somin一闷习On五n)单核细胞中HSFI体含量与IL一lp和TNF一amRNA含量的变化之间呈负相关(r二的三聚 一0.963，r=一0.970，P<0.01)。结论.IJS发热时单核细胞中HSFI活性三聚体含量增加。.IJS发热时单核细胞中HSFI活性三聚体含量与单核细胞内几一lp、TNF一以加丑润A表达量之间呈负相关关系。3 .IJS诱导发热时单核细胞中HSFI可能通过抑制xL一lp、TNF一a等内生性致热原基因的表达而限制体温升高。更多还原

【Abstract】 IntroductionHeat shock factor (HSF) is generally found in the eukaryotic cell, which is high conserved in the structure and function. HSFl is sensitive to all kinds stresses. HSFl transforms form inactivated monomer to activated trimerization that steps into the nucleolus and combines with the heat shock element ( HSE) , which lies in the upstream gene of protein 70 ( HSP70 ) , to promote HSP70 transcription and expression.Studies indicate that many inflammatory stimulators invade the body and activate monocyte to produce endogenous pydogen ( EP) , including interleukin-1 (IL-1) , tumor nectosis factor, which is the important key to induce fever.The promoter of IL-1 (3 gene has an integrate HSE sequence, and theTNF-agene near the promoter exists many incomplete HSE sequences. Consequently, it’s deduced that activated HSFlont only conbines with the HSE sequence of HSP70, but also with IL-1 TNF-, and affects their transcriptions, there are few reports about whether HSFl regulates the temperature by affecting the expression of IL-l,TNF-during fever.Our study aim at the effects of HSFl expression on the level of the gene transcription of IL-1 ,TNF-a. We study the expression of HSFl and IL-13 , TNF-amRNA in monocyte and their relationship during different fever stages in rabbit fever model induced by LPS. This study is very critical to identify the mechanism of thermo-limitation.Methods1. Animol model preparation; male rabbits were used by their weight from 2.0kg to 3. Okg, provided by animal department. They were divided into sixgroups; the control group, LPS-feverish-80min, LPS- feverish-160min, LPS-fe-verish-240min, LPS- feverish-320min, UPS- feverish-400min group.2. IL-1,TNF-mRNA expression : RT-PCR assay3. HSF1 protein expression ; western blot ( protein immunoblotting)4. image analysis technique and data processingRESULTS1. The body temperature change: Intravenous injection of LPS (0.5 g/kg) produced the double-phase fever in rabbits. The body temperature increases to the first peak value at 60min-phase alter injecting LPS, and to the second peak value at 180min-phase.2. HSF1 expression in the monocyte: The total content of HSF1 (including monomer and trimerization) wasnt obviously change in all groups (p >0. 05) , but the content of HSF1 trimerization increased with the body temperature rise (p<0.01) .3. IL-1,KTNF-mRNA expression in the monocyte at the different phases during the fever: the expression of IL-1mRNA in the monocyte increased at 80min-phase, arrived at the peak at 160min-phase, decreased to the normal level at the 400min-phase . There were significant differences among all groups ( p<0. 01 ). TNF-amRNA production in the monocyte reached the peak value at 80min-phase, then decreased gradually and arrived at the normal level at 320min-phase. There were significant differences among all groups (p <0. 01)4. The relation between the content of HSF1 expression and the content of the IL-1 ,TNF-amRNA production in the monocyte; The relative analysis indicated that in the LPS-feverish rabbit model, the content of the trimerization of HSF1 manifested the negative correlation with the content of the IL-1, TNF-amRNA productions (r= -0.963, r= -0.970; p<0.01).CONCLUSIONS1. The content of the trimerization of HSF1 increased in the monocyte during the LPS-feverish rabbits.2. The content of the trimerization of HSF1 in the monocyte manifested the negative correlation with the content of the IL-1β,TNF-αmRNA productions.3. During the UPS-induced fever, it was possible that HSF1 limited the rise of the body temperature through repressing the gene expressions of the endogery pyrogen-IL-1β,TNF-α.更多还原