本文關(guān)鍵詞： 內(nèi)毒素耐受 高遷移率族蛋白 IRAK-M NF-κB 膿毒癥　出處：《中南大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：背景：高遷移率族蛋白1(HMGB1)生理狀態(tài)下以非組蛋白的形式存在于細(xì)胞核中,可在炎癥反應(yīng)晚期釋放至胞外發(fā)揮炎癥因子的作用。外源性重組HMGB1可以引起內(nèi)毒素耐受。內(nèi)毒素耐受是機(jī)體抑制自身炎癥反應(yīng)過度發(fā)展的有效手段,表現(xiàn)為抑制TNF-α等促炎因子的表達(dá)而并不抑制IL-10等抑炎因子的表達(dá)。雖然內(nèi)毒素耐受能在一定程度上抑制炎癥反應(yīng)的泛濫,但若任其發(fā)展,便會削弱機(jī)體的防御能力,增加二次感染甚至死亡的幾率。目前有關(guān)內(nèi)毒素耐受機(jī)制的研究尚無定論,我們不知道該過程通過什么來介導(dǎo),是否存在關(guān)鍵的啟動因子。 目的：研究經(jīng)典內(nèi)毒素耐受(小劑量LPS引起)的發(fā)生機(jī)制,說明內(nèi)源性HMGB1在其中不可替代的獨特作用,并闡明其機(jī)制。 方法：課題分為四個部分。第一章,應(yīng)用重組HMGB1或經(jīng)熱處理的重組HMGB1預(yù)處理小鼠1小時后,給予其大劑量LPS打擊, ELISA檢測血清中TNF-α的含量,WB及免疫組織化學(xué)染色法檢測肝臟中其中IRAK-M的表達(dá)；第二章,應(yīng)用小劑量LPS預(yù)處理小鼠24小時后,給予其大劑量LPS打擊,ELISA檢測血清中HMGB1的含量,WB檢測肝臟中HMGB1的表達(dá)；應(yīng)用小劑量LPS預(yù)處理RAW264.7細(xì)胞18小時后,給予其大劑量LPS打擊,免疫熒光染色觀察HMGB1的核-胞漿轉(zhuǎn)移情況；第三章,應(yīng)用小劑量LPS預(yù)處理小鼠,0.5,4和10小時后分別連續(xù)腹腔注射正丁酸鈉,24小時后腹腔注射大劑量LPS, RT-PCR檢測肝臟中TNG-α mRNA的表達(dá),WB檢測肝臟中HMGB1的表達(dá),ELISA檢測血清中TNF-α的含量；第四章,應(yīng)用小劑量LPS預(yù)處理小鼠,2,12,22小時后分別連續(xù)腹腔注射HMGB1中和抗體,24小時后腹腔注射大劑量LPS, ELISA檢測血清中TNF-α的含量,WB檢測肝臟中IRAK-M的表達(dá),WB檢測NF-κB在肝臟胞核中的表達(dá),EMSA檢測肝臟中NF-κB與DNA的結(jié)合能力。 結(jié)果：第一章,重組HMGB1預(yù)處理1小時可以引起內(nèi)毒素耐受,其機(jī)制與上調(diào)負(fù)性調(diào)控蛋白IRAK-M的表達(dá)有關(guān),與混在其中的微量LPS無關(guān)；第二章：小劑量LPS預(yù)處理24小時可以引起內(nèi)源性HMGB1表達(dá)及釋放的增加；第三章,HMGB1抑制劑正丁酸鈉可以抑制小劑量LPS引起的內(nèi)毒素耐受；第四章,HMGB1中和抗體可以通過下調(diào)IRAK-M的表達(dá),恢復(fù)NF-κB的活性來阻礙LPS引起的內(nèi)毒素耐受現(xiàn)象。 結(jié)論：小劑量LPS引起的內(nèi)毒素耐受現(xiàn)象需要內(nèi)源性HMGB1的參與,其機(jī)制與上調(diào)IRAK-M的表達(dá),抑制NF-κB通路的活性有關(guān)。
[Abstract]:Background: high mobility group protein 1 (HMGB1) under physiological condition in the form of non histone proteins in the nucleus, can play the role of inflammatory cytokines in inflammatory reaction later released into the extracellular. Exogenous HMGB1 can induce endotoxin tolerance. Endotoxin tolerance is the body itself inhibiting inflammation effectively means excessive development. Expression of TNF- alpha inhibits the expression of pro-inflammatory cytokines and anti-inflammatory cytokines does not inhibit IL-10. Although the spread of endotoxin tolerance can inhibit inflammatory reaction in a certain extent, but if unchecked, will weaken the body's defenses, increasing two times of infection and death risk. At present the research on the mechanism of endotoxin tolerance there is no conclusive, we do not know the process mediated by what, if there is the key factor to start.
Objective: To study the mechanism of classical endotoxin tolerance (small dose of LPS), and to illustrate the unique role of endogenous HMGB1 in its irreplaceable and elucidate its mechanism.
Methods: the subject is divided into four parts. The first chapter, the application of recombinant HMGB1 or heat-treated recombinant HMGB1 pretreatment of mice after 1 hours, given large doses of LPS against TNF- content alpha ELISA in serum, WB and immunohistochemical staining in liver IRAK-M expression; the second chapter. Pretreatment of mice 24 hours after the application of small dose of LPS, given the high dose of LPS attack, HMGB1 content of ELISA in serum, the expression of HMGB1 WB detected in the liver; application of low dose LPS pretreatment of RAW264.7 cells after 18 hours, given its large dose LPS blow, immunofluorescence staining to observe HMGB1 nucleus - cytoplasm transfer; the third chapter, the pretreatment of mice with small dose of LPS, 0.5,4 and 10 hours respectively after continuous intraperitoneal injection of sodium butyrate, 24 hours after intraperitoneal injection of high-dose LPS, RT-PCR to detect the expression of TNG- in liver alpha mRNA, HMGB1 WB detection of liver in table As the content of TNF-, alpha ELISA in serum; the fourth chapter, the pretreatment of mice with small dose LPS and 2,12,22 hours respectively after intraperitoneal injection of HMGB1 neutralizing antibody, 24 hours after intraperitoneal injection of high-dose LPS, TNF- content of alpha ELISA in serum, the expression of IRAK-M WB detected in the liver, to detect the expression of WB kappa NF- B in the liver cell nucleus. The binding ability of NF- kappa B and DNA EMSA detection in the liver.
Results: in the first chapter, the recombinant HMGB1 was pretreated for 1 hours can induce endotoxin tolerance, expression mechanism and increase the negative regulatory protein IRAK-M, and not in the mixed trace LPS; the second chapter: low dose LPS pretreatment for 24 hours can be caused by endogenous HMGB1 expression and release increased; the third chapter, endotoxin tolerance HMGB1 inhibitors can inhibit the sodium butyrate induced by low dose of LPS; the fourth chapter, HMGB1 neutralizing antibody can downregulate the expression of IRAK-M, recovery of NF- kappa B activity prevents endotoxin tolerance induced by LPS.
Conclusion: the phenomenon of endotoxin tolerance induced by small dose of LPS requires the participation of endogenous HMGB1, and its mechanism is related to the up-regulation of IRAK-M expression and the inhibition of the activity of NF- kappa B pathway.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R631

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本文編號：1542194