本文選題：熱休克因子1 + 精氨酸加壓素　； 參考：《中國(guó)醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 前言 恒溫動(dòng)物和人在發(fā)熱時(shí)體溫很少超過一定限度，例如，人體體溫很少超過41℃，稱為熱限現(xiàn)象，提示體內(nèi)存在著調(diào)控體溫的自限機(jī)構(gòu)，但其機(jī)制尚不十分清楚。脂多糖(lipopolysaccharide，LPS)作為外源性致熱原可致體溫升高。精氨酸加壓素(arginine vasopressin，AVP)是一種內(nèi)源性解熱物質(zhì)，主要作用于腹側(cè)中隔區(qū)(vent ral septal area，VSA)。有報(bào)道向VSA注射AVP能顯著地抑制家兔的內(nèi)毒素(endotoxin，ET)性發(fā)熱反應(yīng)。 熱休克因子(Heat shock transcription factor，HSF)是一組結(jié)構(gòu)和功能具有廣泛同源性，普遍存在于真核生物細(xì)胞中的核蛋白，包括HSF1、HSF2、HSF3、HSF4。其中HSF1對(duì)溫度刺激敏感，被激活時(shí)由單體結(jié)合成三聚體，具有調(diào)節(jié)熱休克蛋白(Heat shock protein，HSP)及多種生物活性物質(zhì)轉(zhuǎn)錄的功能。本研究室以往的研究結(jié)果顯示，HSF1能夠抑制單核細(xì)胞內(nèi)生性致熱原的產(chǎn)生及中樞發(fā)熱介質(zhì)的產(chǎn)生。但是HSF1的激活是否能夠調(diào)控中樞AVP的水平，進(jìn)而，AVP發(fā)揮對(duì)體溫的負(fù)調(diào)節(jié)作用，相關(guān)的研究還未見報(bào)道。 本實(shí)驗(yàn)通過復(fù)制家兔LPS發(fā)熱模型，觀察發(fā)熱誘導(dǎo)的HSF1聚合對(duì)發(fā)熱過程及腦內(nèi)AVP含量的影響，并通過中樞給予槲皮素(quercetin，QCT)抑制HSF1的活化，旨在進(jìn)一步探討發(fā)熱時(shí)HSF1限控體溫的相關(guān)中樞調(diào)節(jié)機(jī)制。 材料與方法 1、動(dòng)物分組、模型及標(biāo)本制備 (1)動(dòng)物分組健康雄性新西蘭白兔70只，隨機(jī)分為四組：正常對(duì)照組(N)、槲皮素組(Q)、脂多糖組(L)和槲皮素+脂多糖組(Q+L)。 (2)腦室插管在應(yīng)用1％戊巴比妥鈉(3 ml·kg~(-1))靜脈麻醉下，將動(dòng)物固定在SN-2型腦立體定位儀上。按Sawyer氏兔腦圖譜，在P1L4-5H5(側(cè)腦室)處埋藏一外徑為1mm的塑料管，用牙科水泥固定，術(shù)后將管端中封閉。術(shù)后家兔單籠飼養(yǎng)，常規(guī)喂養(yǎng)1wk后進(jìn)行實(shí)驗(yàn)。 (3)復(fù)制發(fā)熱模型用無熱原的生理鹽水將精制的大腸桿菌內(nèi)毒素-LPS稀釋成0.5μg·ml~(-1)，按1μg·kg~(-1)體重，經(jīng)家兔耳緣靜脈注射。 (4)取材N組和Q組于測(cè)溫后60 min處死家兔，其它兩組分別在給予LPS后0、30、60、180、240和360 min時(shí)處死家兔，迅速取出家兔下丘腦及腹中隔區(qū)，取一部分放入生理鹽水中，煮沸5 min。制備組織勻漿，離心后取上清置-20℃保存待進(jìn)行AVP含量的測(cè)定；另取一部分迅速放入液氮中，20 min后置-70℃深度冰箱中保存，待進(jìn)行HSF1和HSP70表達(dá)量的測(cè)定。 2、檢測(cè)指標(biāo) 實(shí)時(shí)監(jiān)測(cè)體溫的變化，體溫變化情況采用△T，TRI進(jìn)行分析；應(yīng)用Western印跡法檢測(cè)不同實(shí)驗(yàn)條件下下丘腦HSF1和HSP70的表達(dá)；用放射免疫法檢測(cè)下丘腦及腹中隔區(qū)AVP含量的變化。 3、數(shù)據(jù)分析 所有實(shí)驗(yàn)數(shù)據(jù)均用均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示，采用SPSS11.0軟件統(tǒng)計(jì)分析，相關(guān)分析用相關(guān)性檢驗(yàn)分析法。 實(shí)驗(yàn)結(jié)果 1、家兔的體溫變化 家兔靜脈注射LPS(1μg·kg~(-1))后引起雙峰熱。即60 min時(shí)體溫上升形成第一個(gè)高峰，體溫變化值為1.32±0.24℃。在180 min時(shí)，體溫上升形成第二個(gè)高峰，體溫變化值為1.84±0.35℃。各組體溫變化最大值(△Tmax)由低至高順序?yàn)椋篞組＜N組＜L組＜Q+L組；其中，Q+L組與L組比較，在給予LPS后240～360min期間，△T及TRI_6均呈顯著性增高(P＜0.05)，且可見發(fā)熱時(shí)程延長(zhǎng)。 2、下丘腦及VSA中AVP的含量變化 各組VSA中的AVP含量均呈現(xiàn)隨發(fā)熱幅度增大而增加，二者之間呈明顯的正相關(guān)(r=0.913，P＜0.01)。Q+L組與L組比較，VSA中的AVP含量顯著增高(240～360 min)(P＜0.05)，但下丘腦中AVP的含量均無顯著變化(P＞0.05)。 3、下丘腦及腹中隔區(qū)HSF1和HSP70的表達(dá) 在LPS致熱過程中，可見從給予LPS后60 min(△T：1.32±0.24℃)起HSF1三聚體的表達(dá)顯著增高，達(dá)到體溫最高值后隨著HSF1三聚體的表達(dá)水平的進(jìn)一步升高，，體溫逐漸下降。在各組HSF1的總體含量無顯著變化。應(yīng)用QCT可顯著地抑制HSF1的聚合，同時(shí)可見HSP70表達(dá)水平相應(yīng)降低。 4、HSF1三聚體含量與體溫及AVP含量變化的相關(guān)性分析 LPS致發(fā)熱過程中，在達(dá)到體溫高峰以后(180～360 min)，下丘腦中HSF1三聚體的含量與體溫變化呈負(fù)相關(guān)(r=-0.897，P＜0.01)；與VSA中的AVP含量也呈負(fù)相關(guān)(r=-0.774，P＜0.01)。 結(jié)論 1、VSA區(qū)AVP的含量隨發(fā)熱幅度增大而增加。 2、下丘腦HSF1三聚體的形成可一定程度地抑制LPS引起的發(fā)熱反應(yīng)，并可見VSA區(qū)AVP的含量減少。
[Abstract]:Preface
The temperature of the thermostatic animals and people is rarely more than a certain limit, for example, the body temperature is rarely more than 41 degrees C, which is called the heat limit phenomenon, suggesting that there is a self limiting mechanism for regulating body temperature, but its mechanism is not very clear. Lipopolysaccharide (LPS) as an exogenous pyrogen can increase the body temperature. Arginine vasopressin (arginin E vasopressin, AVP) is an endogenous antipyretic substance which is mainly used in the ventral septum (vent ral septal area, VSA). It is reported that the injection of AVP to VSA can significantly inhibit the endotoxin (endotoxin, ET) sex fever reaction in rabbits.
Heat shock factor (Heat shock transcription factor, HSF) is a group of structures and functions that are widely homologous and ubiquitous in eukaryotic cells, including HSF1, HSF2, HSF3, HSF4. in which HSF1 is sensitive to temperature stimuli and is activated by a monomer, which regulates heat shock proteins (Heat shock) and Previous studies in this study show that HSF1 can inhibit the production of endogenous pyrogen and the production of central fever medium in monocyte, but whether activation of HSF1 can regulate the level of central AVP, and then AVP plays a negative regulatory role on body temperature, and the related research has not been reported.
In this experiment, the effect of fever induced HSF1 polymerization on the fever process and the AVP content in the brain was observed by replicating the rabbit LPS fever model, and the activation of HSF1 was inhibited by the central administration of quercetin (quercetin, QCT). The purpose of this study was to further explore the central regulatory mechanism of HSF1 limited temperature controlled by HSF1 in fever.
Materials and methods
1, animal grouping, model and specimen preparation
(1) 70 healthy male New Zealand white rabbits were randomly divided into four groups: normal control group (N), quercetin group (Q), lipopolysaccharide group (L) and quercetin + lipopolysaccharide group (Q+L).
(2) intraventricular intubation in the use of 1% pentobarbital sodium (3 ml. Kg~ (-1)) intravenous anesthesia, the animals were fixed on the SN-2 stereotaxis. According to the Sawyer rabbit brain atlas, a plastic tube with a external diameter of 1mm was buried in the P1L4-5H5 (lateral ventricle), fixed with dental cement and closed in the end of the tube after operation. The rabbits were fed in a single cage after the operation, and the 1wk was fed routinely after the operation. Do the experiment.
(3) the replicating fever model diluted the refined Escherichia Coli Endotoxin -LPS into 0.5 mu g / ml~ (-1) with the physiological saline of pyrogen, and injected the rabbit ear vein with 1 mu g. Kg~ (-1) body weight.
(4) the rabbits were killed in group N and group Q at 60 min after temperature measurement. The other two groups were killed at 0,30,60180240 and 360 min after LPS, and the rabbit hypothalamus and ventral septum were removed quickly. Some of them were put into the normal saline, and the tissue homogenate was prepared by boiling 5 min., and then collected at -20 C after centrifugation to determine the AVP content. Some of them were quickly placed in liquid nitrogen and stored in 20 min deep refrigerators at -70 C depth. The expression levels of HSF1 and HSP70 were determined.
2, detection index
The changes of body temperature were monitored in real time. The changes of body temperature were analyzed with delta T and TRI, and the expression of HSF1 and HSP70 in hypothalamus under different experimental conditions was detected by Western blotting, and the changes in the content of AVP in the hypothalamus and ventral septum were detected by radioimmunoassay.
3, data analysis
All the experimental data were expressed by mean + standard deviation ((+) s), and SPSS11.0 software was used for statistical analysis. Correlation analysis was conducted by correlation test method.
experimental result
1, the change of body temperature in rabbits
After intravenous injection of LPS (1 mu g. Kg~ (-1)), the heat of Shuangfeng was caused by the increase of temperature at 60 min. The temperature change was 1.32 + 0.24 C. At 180 min, the temperature rise formed second peaks, the temperature change was 1.84 + 0.35 degrees. The maximum temperature change of each group (delta Tmax) was from low to high in order of Q group < N < L < Q+L. In group Q+L, T and TRI_6 were significantly increased during the period from 240 to 360min after giving LPS, compared with group L (P < 0.05), and the duration of fever was longer.
2, changes in the content of AVP in the hypothalamus and VSA
The content of AVP in each group of VSA increased with the increase of the amplitude of fever, and there was a significant positive correlation between the two (r=0.913, P < 0.01). Compared with the L group,.Q+L group increased significantly (240~360 min) in VSA (P < 0.05), but the AVP content in the hypothalamus was not significantly changed (P > 0.05).
3, expression of HSF1 and HSP70 in the hypothalamus and ventral septum
During the heat process of LPS, the expression of HSF1 trimer increased significantly from the 60 min (delta T:1.32 + 0.24 C) after LPS, and the body temperature decreased gradually with the increase of the expression level of HSF1 trimer after the highest temperature of the body temperature. The total content of HSF1 in each group was not significantly changed. The application of QCT could significantly inhibit the polymerization of HSF1, at the same time The expression level of HSP70 was reduced accordingly.
4, the correlation between the content of HSF1 trimer and the changes of body temperature and AVP content
In the process of fever induced by LPS, after reaching the peak of temperature (180~360 min), the content of HSF1 trimer in the hypothalamus was negatively correlated with the change of body temperature (r=-0.897, P < 0.01), and negatively correlated with the AVP content in VSA (r=-0.774, P < 0.01).
conclusion
1, the content of AVP in VSA zone increases with the increase of heating rate.
2, the formation of hypothalamus HSF1 trimer can inhibit the fever reaction induced by LPS to some extent, and the content of AVP in VSA region can be reduced.

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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