高迁移率族蛋白1在内毒素耐受中的作用及其机制研究

发布时间：2018-02-27 10:00

本文关键词： 内毒素耐受高迁移率族蛋白 IRAK-M NF-κB 脓毒症　出处：《中南大学》2013年博士论文　论文类型：学位论文

【摘要】：背景：高迁移率族蛋白1(HMGB1)生理状态下以非组蛋白的形式存在于细胞核中,可在炎症反应晚期释放至胞外发挥炎症因子的作用。外源性重组HMGB1可以引起内毒素耐受。内毒素耐受是机体抑制自身炎症反应过度发展的有效手段,表现为抑制TNF-α等促炎因子的表达而并不抑制IL-10等抑炎因子的表达。虽然内毒素耐受能在一定程度上抑制炎症反应的泛滥,但若任其发展,便会削弱机体的防御能力,增加二次感染甚至死亡的几率。目前有关内毒素耐受机制的研究尚无定论,我们不知道该过程通过什么来介导,是否存在关键的启动因子。目的：研究经典内毒素耐受(小剂量LPS引起)的发生机制,说明内源性HMGB1在其中不可替代的独特作用,并阐明其机制。方法：课题分为四个部分。第一章,应用重组HMGB1或经热处理的重组HMGB1预处理小鼠1小时后,给予其大剂量LPS打击, ELISA检测血清中TNF-α的含量,WB及免疫组织化学染色法检测肝脏中其中IRAK-M的表达；第二章,应用小剂量LPS预处理小鼠24小时后,给予其大剂量LPS打击,ELISA检测血清中HMGB1的含量,WB检测肝脏中HMGB1的表达；应用小剂量LPS预处理RAW264.7细胞18小时后,给予其大剂量LPS打击,免疫荧光染色观察HMGB1的核-胞浆转移情况；第三章,应用小剂量LPS预处理小鼠,0.5,4和10小时后分别连续腹腔注射正丁酸钠,24小时后腹腔注射大剂量LPS, RT-PCR检测肝脏中TNG-α mRNA的表达,WB检测肝脏中HMGB1的表达,ELISA检测血清中TNF-α的含量；第四章,应用小剂量LPS预处理小鼠,2,12,22小时后分别连续腹腔注射HMGB1中和抗体,24小时后腹腔注射大剂量LPS, ELISA检测血清中TNF-α的含量,WB检测肝脏中IRAK-M的表达,WB检测NF-κB在肝脏胞核中的表达,EMSA检测肝脏中NF-κB与DNA的结合能力。结果：第一章,重组HMGB1预处理1小时可以引起内毒素耐受,其机制与上调负性调控蛋白IRAK-M的表达有关,与混在其中的微量LPS无关；第二章：小剂量LPS预处理24小时可以引起内源性HMGB1表达及释放的增加；第三章,HMGB1抑制剂正丁酸钠可以抑制小剂量LPS引起的内毒素耐受；第四章,HMGB1中和抗体可以通过下调IRAK-M的表达,恢复NF-κB的活性来阻碍LPS引起的内毒素耐受现象。结论：小剂量LPS引起的内毒素耐受现象需要内源性HMGB1的参与,其机制与上调IRAK-M的表达,抑制NF-κB通路的活性有关。
[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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R631

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