本文關鍵詞： 高遷移率族蛋白B1 慢加急性肝功能衰竭 HMGB1單克隆抗體 Toll樣受體 腫瘤壞死因子 吡咯烷二硫基甲酸鹽 慢加急性肝功能衰竭 高遷移率族蛋白B1　出處：《武漢大學》2014年博士論文　論文類型：學位論文

【摘要】：第一部分：阻斷 HMGB1對慢加急性肝功能衰竭大鼠的影響 目的：慢加急性肝功能衰竭是一種以肝功能損害,以及肝細胞大量壞死為核心癥狀嚴重疾病,具有很高的死亡率。HMGB1是近年來發(fā)現(xiàn)的一種強大的炎癥調節(jié)因子。本研究應用HMGB1單克隆抗治療慢加急性肝功能衰竭大鼠,觀察HMGB1單克隆抗體對慢加急性肝功能衰竭大鼠的保護作用。 方法：大鼠慢加急性肝功能衰竭模型由人血白蛋白,D—氨基半乳糖以及LPS聯(lián)合誘導。大鼠被隨機分為3組,正常組,模型組以及HMGB1單克隆抗體治療組。HMGB1單克隆抗體治療組大鼠在模型誘導后12小時及24小時時間點由尾靜脈注射HMGB1單克隆抗體200μg/kg,正常組及模型組注射等劑量的生理鹽水作為對照。采用鱟試劑檢測各組大鼠血清內毒素水平,應用ELISA方法檢測了血清HMGB1, TNF-α, IFN-γ水平,同時檢測了各組大鼠肝臟組織學及肝細胞凋亡情況,并且應用Western b1ot方法檢測了各組大鼠肝織中HMGB1, TLR4和P65的表達情況。 結果：HMGB1單克隆抗體能夠明顯降低血清中內毒素,HMGB1, TNF-α, IFN-γ水平,改善肝組織的病理損害,減少肝細胞的凋亡,同時下調肝組織中HMGB1,TLR4, P65的表達量。 結論：HMGB1單克隆抗體對慢加急性肝功能衰竭大鼠具有保護作用,即使是在模型誘導后24小時仍然有效。此效應可能與TLR4信號途徑相關。 第二部分：TLR4單克隆抗體對慢加急肝功能衰竭大鼠的影響 目的：慢加急性肝功能衰竭是臨床上常見危重癥之一,TLR4信號途徑及炎癥因子HMGB1在其發(fā)病過程中發(fā)揮重要作用。本研究采用TLR4單克隆抗體對慢加急性肝功能衰竭大鼠進行治療,觀察TLR4信號途徑被阻斷后對慢加急性肝功能衰竭大鼠及炎癥因子HMGB1的影響。 方法：將32只雄性Wistar大鼠隨機分為正常組、模型組、IgG對照組及TLR4單克隆抗體治療組。除正常組大鼠外,其余每組大鼠采用人血白蛋白,D-氨基半乳糖以及LPS聯(lián)合誘導的大鼠慢加急性肝功能衰竭模型。IgG對照組大鼠在急性攻擊24小時后,予以尾靜脈注射兔抗鼠IgG100μg/kg作為對照。TLR4單克隆抗體治療組大鼠在急性攻擊24小時后,予以尾靜脈注射兔抗鼠TLR4單克隆抗體100μg/kg。模型組大鼠尾靜脈注射等量的生理鹽水作為對照。觀察TLR4單克隆抗體對大鼠肝臟組織學、血清ALI、TNF-α、IFN-γ和HMGB1水平以及肝組織中HMGB1水平的影響。 結果：與正常組相比,模型組大鼠肝組織明顯破壞,炎癥細胞浸潤明顯,血清中ALT、TNF-α、IFN-γ和HMGB1水平明顯升高(P0.05)。TLR4單克隆抗體能夠明顯改善慢加急性肝功能衰竭大鼠肝臟組織病理學損害,降低血清中ALT、TNF-a、IFN-γ和HMGB1水平,并且降低肝組織中HMGB1水平(P0.05)。而與模型組相比,以上指標在IgG對照組中無明顯差異(P0.05)。 結論：TLR4單克隆抗體能夠保護慢加急性肝功能衰竭大鼠并減少HMGB1胞外釋放以及肝組織中HMGB1的產(chǎn)生。TLR4單克隆抗體,慢加急性肝功能衰竭,高遷移率族蛋白B1 第三部分：阻斷NF-κB和TNF-α對慢加急性肝功能衰竭大鼠的影響 目的：在慢加急性肝功能衰竭的發(fā)病機制中,炎癥介質HMGB1發(fā)揮著重要作用。而HMGB1主要通過TLR4-NF-κB信號通路在發(fā)揮其作用。而在TLR4-NF-κB信號通路中,NF-κB和TNF-α又是兩個處于中心地位的炎癥因子。本研究的目的在于探索NF-κB和TNF-α被阻斷后對于慢加急性肝功能衰竭大鼠的影響。 方法：本研究應用人血白蛋白,D一氨基半乳糖及脂多糖聯(lián)合誘導大鼠慢加急性肝功能衰竭模型,并采用TNF-α多克隆抗體(TNF-α抑制劑)及吡咯烷二硫基甲酸鹽(PDTC, NF-κB抑制劑)治療肝功能衰竭大鼠,觀察TNF-α多克隆抗體和PDTC對慢加急性肝功能衰竭大鼠的影響。本研究進一步采用PMA誘導U937細胞成為巨噬細胞,并應用LPS刺激,觀察了PDTC對巨噬細胞的影響。 結果：TNF-α和NF-κB抑制劑都能夠有效降低肝臟和外周血中HMGB1水平以及血清中炎癥因子的水平,并能夠抑制TLR4-NF-KB信號通路。TNF-α抑制劑還能夠延長慢加急性肝功能衰竭大鼠的存活時間提高生存率,相比之下,NF-κB抑制劑PDTC卻加速了慢加急性肝功能衰竭大鼠的死亡。為了進一步研究PDTC在炎癥反應中的作用,我們進一步檢測了PDTC在U937細胞中的作用,結果顯示PDTC能夠提高U937細胞的活性,并且降低HMGB1水平以及血清中炎癥因子的水平,并能夠抑制TLR4-NF-κB信號通路。 結論：TNF-α抗體和PDTC都能夠抑制TLR4-NF-κB信號通路的激活,減少炎癥因子的釋放,抑制炎癥反應。TNF-α抗體對慢加急性肝功能衰竭大鼠具有保護作用,而PDTC不能保護肝功能衰竭大鼠。
[Abstract]:Part one: blocking
Effect of HMGB1 on rats with chronic acute liver failure
Objective: acute on chronic liver failure is a damage to the liver function, and massive necrosis of liver cells as the core symptoms of the serious disease, with high mortality of.HMGB1 is a powerful inflammatory regulatory factor found in recent years. In this study, using HMGB1 monoclonal antibody treatment of acute on chronic liver failure in rats, to observe the protection effect of HMGB1 monoclonal antibody in acute on chronic liver failure in rats.
Methods: the rat model of acute on chronic liver failure by Human Albumin, D and LPS induced by D-galactosamine. Rats were randomly divided into 3 groups, normal group, model group and HMGB1 treatment group.HMGB1 monoclonal antibody monoclonal antibody treated rats in the model induced after 12 hours and 24 hours from the time point intravenous injection of HMGB1 monoclonal antibody 200 g/kg, normal saline group and model group injected dose by limulus reagent as control. The serum levels of endotoxin, ELISA method was used to detect serum HMGB1, TNF- alpha, gamma IFN- level were detected and liver cell apoptosis in liver tissue was the rat, and used Western b1ot method to detect the liver of rats in the fabric HMGB1, expression of TLR4 and P65.
Results: HMGB1 monoclonal antibody could significantly reduce the levels of endotoxin, HMGB1, TNF-, IFN-, and improve the pathological damage of liver tissue, reduce the apoptosis of liver cells, and down regulate the expression of HMGB1, TLR4 and P65 in liver tissue.
Conclusion: HMGB1 monoclonal antibody has protective effect on rats with acute on chronic liver failure, even after 24 hours of induction. This effect may be related to TLR4 signaling pathway.
The second part: the effect of TLR4 monoclonal antibody on rats with chronic acute liver failure
Objective: acute on chronic liver failure is one of the common clinical critical disease, TLR4 signal pathway and inflammatory factor HMGB1 play an important role in its pathogenesis. This study used in acute on chronic liver failure rats were treated with TLR4 monoclonal antibody, observe the effect of acute on chronic liver failure rats and inflammatory factors HMGB1 is blocking TLR4 signaling.
Methods: 32 male Wistar rats were randomly divided into normal group, model group, IgG control group and TLR4 monoclonal antibody treatment group. In addition to the normal group rats, the rats in each group by Human Albumin, D- and LPS induced by D-galactosamine in rats with chronic model of acute liver failure.IgG control group rats in the acute attack after 24 hours of intravenous injection of Rabbit anti rat IgG100 monoclonal antibody g/kg as the control.TLR4 treated rats in the acute attack after 24 hours of saline to intravenous injection of Rabbit anti rat TLR4 monoclonal antibody 100 g/kg. model rats tail vein injected as control. Observe TLR4 monoclonal antibody to rat liver tissue, serum ALI, TNF- alpha, gamma and IFN- affect the level of HMGB1 and HMGB1 level in liver tissue.
Results: compared with normal group, the liver tissue of model rats obviously damage, inflammatory cell infiltration, serum ALT, TNF- alpha, IFN- gamma and HMGB1 levels were significantly higher (P0.05).TLR4 monoclonal antibody can significantly improve the pathological liver tissue of rats with acute on chronic liver failure damage, reduce the levels of ALT, TNF-a IFN-, gamma and HMGB1 levels, and reduce the level of HMGB1 in liver tissue (P0.05). Compared with the model group, the above indexes had no significant difference in the IgG control group (P0.05).
Conclusion: TLR4 McAb can protect rats from chronic acute liver failure and reduce HMGB1 extracellular release and the production of HMGB1 in liver tissue,.TLR4 monoclonal antibody, slow acute liver failure, high mobility group protein B1.
The third part: the effect of blocking NF- kappa B and TNF- alpha on rats with chronic acute liver failure
Objective: in the pathogenesis of acute on chronic liver failure, inflammatory mediators play an important role. HMGB1 and HMGB1 mainly through the TLR4-NF- B pathway to play its role in the TLR4-NF- B pathway, NF- kappa B and TNF- alpha is two at the center of the inflammatory factor. The purpose of this study is to explore the effect of acute on chronic liver failure rats NF- kappa B alpha and TNF- was blocked.
Methods: This study used Human Albumin D, a D-galactosamine and lipopolysaccharide induced rat acute on chronic liver failure model, and using TNF- alpha polyclonal antibody (TNF- alpha inhibitor) and pyrrolidine two Sbased formate (PDTC, NF- kappa B inhibitor) treatment of hepatic failure in rats, observe the effect alpha TNF- polyclonal antibody and PDTC in acute on chronic liver failure rats. This study further by PMA induced U937 cells into macrophages, and the application of LPS stimulation, the effect of PDTC on macrophages.
Results: TNF- and NF- alpha kappa B inhibitor can effectively reduce liver and peripheral blood HMGB1 levels and serum levels of inflammatory factors, and TLR4-NF-KB signal pathway of.TNF- alpha inhibitors could also prolong acute on chronic liver failure rats survival time and improve the survival rate, compared with NF- kappa B inhibitor PDTC it accelerates ACLF death in rats. In order to further study the role of PDTC in inflammation, we further examined the role of PDTC in U937 cells, the result shows that PDTC can improve the activity of U937 cells, and decrease the level of HMGB1 and serum levels of inflammatory factors, which can inhibit the expression of TLR4-NF- the B signaling pathway.
Conclusion: both TNF- alpha antibody and PDTC can inhibit the activation of TLR4-NF- kappa B signaling pathway, reduce the release of inflammatory factors and inhibit the inflammatory response..TNF- alpha antibody has protective effects on rats with chronic acute liver failure, while PDTC can not protect liver failure rats.

【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R575.3

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