【作者】 李旭宏；

【导师】 龚建平；

【作者基本信息】 重庆医科大学 ， 外科学， 2005， 博士

【摘要】 脓毒症(sepsis)至今仍是临床治疗中的难点,其导致的死亡率高达 30-40%,而在所有脓毒症病例中,由革兰氏阴性(G-)细菌感染引起者约占 60%。内毒素又名脂多糖(lipopolysaccharide, LPS),是 G-细菌细胞壁上的主要成分,也是 G-细菌最为重要的致病物质,它能激活单核-巨噬细胞系统(mononuclear phagocytic system, MPS),引起TNF-α 等大量前炎症介质释放,导致全身炎症反应综合征(systemic inflammatory response syndrome, SIRS)及器官损害。 然而,机体在遭受LPS刺激的同时也能产生相应的保护机制——即内毒素耐受(endotoxin tolerance)状态。所谓内毒素耐受是指:机体或单核-巨噬细胞经LPS预处理后,对LPS的再次刺激呈低反应性。目前认为,内毒素耐受是生物在长期进化过程中形成的一种保守的负反馈调节机制,是一种适应性应答,能避免了机体对LPS刺激的持续过度反应,是机体防御机制的重要组成部分。 最近的研究表明,白介素1受体相关激酶-M(IRAK-M)对LPS信号通路起负性调节作用,从而有可能参与内毒素耐受的形成机制。本研究通过建立内毒素耐受的小鼠及Kupffer细胞模型,观察在内毒毒耐受状态下机体及细胞中IRAK-M表达的变化,初步确定IRAK-M与内毒素耐受的关系;进而采用RNA干扰技术在体外沉寂IRAK-M的基因表更多还原

【Abstract】 Today, sepsis is still a nodus in clinical treatment, of which the mortality is approximately 30-40%, and about 60% of cases are caused by Gram-negative bacteria infection. Endotoxin is also named as lipopolysaccharide (LPS), which is the main component in the outer membrane of Gram-negative bacteria, and is the most important pathogenic factor of this kind of bacteria. LPS can activate mononuclear phagocytic system (MPS), leading to the release of multiple pro-inflammatory mediators including TNF-α. As a result, systemic inflammatory response syndrome (SIRS) and organ damage occurrence. However, LPS stimulation can induce a protective mechanism,the endotoxin tolerance. Endotoxin tolerance is defined as a reduced capacity of the host or of cultured monocyte/macrophage to respond to LPS activation following a first exposure to this stimulus. Now, endotoxin tolerance is known as a negative feedback regulation and an adaptive response, which prevents host from continuous over-responding to LPS stimulation. So endotoxin tolerance is also an important part of host defence. Recent researches indicated that the interleukin-1 receptor-associated kinase (IRAK) family member, IRAK-M, is a negative regulator of LPS signal pathway, and may involve in the mechanisms of endotoxin tolerance. In this study, animal or cell model of endotoxin tolerance were used to investigate the expression change of IRAK-M under endotoxin tolerance condition, and to observe the relationship between them. Furthermore, RNA interference technique was used to silence IRAK-M gene in RAW264.7 cells, through which we attempt to elucidate the role of IRAK-M in the occurrence of endotoxin tolerane and may provide a new target for the clinical treatment of sepsis. Objective: To investigate the gene expression of IRAK-M in the liver of endotoxin tolerant mice, and to explore the relationship between IRAK-M and endotoxin tolerance on host level. Methods: The experimetal mice were divided randomly into NETT group which was pretreated with i.p injection sterile NS and ETT group which was pretreated with i.p injection of 0.5mg/kg LPS. All animals weretreated with i.p injection of 5mg/kg LPS 24h later. The liver samples were collected for observation of histopathological and ultrastructural changes. The levels of plasma TNF-α were measured by Enzyme linked immunosorbent assay (ELISA). The mRNA expression of TNF-α and IRAK-M in the liver was detected by Reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: The liver damage induced by LPS was attenuated in ETT group comparing with NETT group. The ultrastructural changes of hepatocytes and hepatic sinusoid endothelial cells were slighter in ETT group than in NETT group. In addition, the inhibition of Kupffer cell activation was found in ETT group in comparison with NETT group. In both groups, LPS stimulation increased plasma level of TNF-α and its mRNA expression in the liver, but the increase of these parameters was discounted in ETT group, in which the peak values of plasma TNF-α and its mRNA expression were 62% and 65% of NETT group respectively. The intrahepatic expression of IRAK-M mRNA in NETT group could not be detected until 24h after LPS stimulation. Howere, there was slight expression of IRAK-M mRNA in the liver samples of ETT group before second LPS stimulation, and the expression was rapidly upregulated after LPS treatment, peaking at 6h and being about one-fold higher than the level of NETT group at 24h. Conclusion: LPS pretreatment can induce endotoxin tolerance ofmice, which may be associated with intrahepatic IRAK-M expression and the inhibition of Kupffer cell activation. Objective: To investigate the gene expression of IRAK-M in endotoxin tolerant Kupffer cells, and to explore the relationship between IRAK-M and endotoxin tolerance on cellular level. Methods: Kupffer cells were isolated from the mouse liver by in situ collangenase digestion. All cells were divided randomly into NETT group which was pretreated by incubation in DMEM with 10% FBS and ETT grou更多还原