感染状态下问号钩体脂多糖合成、修饰及其调控机制的研究

发布时间：2018-06-10 10:18

本文选题：问号钩端螺旋体 + 单核-巨噬细胞　；参考：《浙江大学》2012年博士论文

【摘要】：背景和立题依据：由问号钩端螺旋体(Leptospira interrogans,简称钩体)感染引起的钩体病是全世界流行的入兽共患传染病。由于该病通过疫水迅速传播,因而是洪涝、地震时重点监测的传染病之一。我国自然灾害频发,且不少地区是钩体病流行疫区。钩体病临床表现复杂多样,但典型症状和体征是黄疸和出血,其中弥漫性肺出血型患者死亡率高达50%以上。然而,问号钩体致病机制至今不明。大量临床病理资料揭示,钩体病患者病变与内毒素(又称脂多糖,lipopolysaccharide, LPS)中毒极为相似,如各脏器广泛、不同程度内毒素中毒样毛细血管及其内皮的病变,部分肺出血型死亡患者内脏中可见微血栓及凝血物质水平下降等。上述资料提示,问号钩体致病性可能与内毒素密切相关。基因组测序结果表明问号钩体赖株基因组中含有一整套的LPS合成、装配及修饰基因,且钩体含有LPS也已得到证明。有文献报道问号钩体感染豚鼠后其LPS多糖部分会发生改变,并且有报道在钩体病葡萄膜炎患者房水中检测出LPS,进一步表明LPS直接参与问号钩体致病过程,且LPS会发生修饰。研究内容及意义：本文通过研究感染细胞后问号钩体LPS合成基因的表达差异、LPS合成量的变化、LPS中脂质A的结构差异、脂质A结构被修饰后其活性的差异、影响LPS合成和脂质A修饰的主要环境因素并初步探究脂质合成和修饰基因的调控机制,以期揭示LPS在问号钩体致病过程中的作用,并为其它革兰阴性致病菌致病的机制研究提供参考依据。研究方法：本文采用小鼠J774A.1和人THP-1单核-巨噬细胞感染模型,采用基因芯片和实时荧光定量RT-PCR技术测定感染细胞后问号钩体LPS合成基因的表达差异；运用鲎试验测定感染细胞后钩体LPS活性变化；运用KDO试验、Purpald试验、SDS-PAGE-银染色手段、HPLC-MS联用技术测定感染细胞后LPS含量变化；采用质谱技术测定感染细胞后脂质A的结构修饰；采用实时荧光定量RT-PCR技术测定影响脂质A合成及修饰基因表达量的环境因素及调节基因；采用抗体封闭和基因敲除技术确定脂质A合成及修饰基因的调节基因；采用抗体封闭和ELISA手段测定不同结构脂质A诱导单核-巨噬细胞分泌炎症因子的活性及识别受体。实验结果：实验结果表明,感染J774A.1和THP-1细胞后问号钩体34个LPS合成基因中分别有13和11个基因表达水平发生变化,其中参与脂质A合成的限速酶基因lpxB和lpxC及脂肪酸转移酶基因lpxD2和htrB上调表达而脂肪酸转移酶基因lpxD1下调表达。感染两种细胞后问号钩体脂多糖活性分别增加1.97和2.09倍,而其数量仅增加1.02和1.06倍。质谱结果表明感染细胞后,问号钩体由感染前含1条肉豆蔻烯酸、1条月桂烯酸、2条棕榈酸和2条月桂酸、m/z=1921的脂质A转变成各有2条月桂酸、肉豆蔻二烯酸和棕榈酸、m/z=1949的脂质A。感染细胞后问号钩体的脂质A(LepLipidA-Infection)促凝活性为感染前(LepLipidA-EMJH)的3.5倍。两种脂质A均诱导人单核-巨噬细胞THP-1产生炎症因子TNF-α、IL-1β及IL-8,但LepLipidA-Infection该活性较强,分别为LepLipidA-EMJH的1.95、1.95和1.69倍；LepLipidA-EMJH仅由THP-1TLR2识别,而LepLipidA-Infection由THP-1细胞TLR2(主要)和TLR4识别。两种脂质A均诱导小鼠单核-巨噬细胞RAW264.7产生炎症因子TNF-α、IL-1β及IL-6,但两种脂质A该活性相近；两种脂质A均能由RAW264.7细胞TLR2(主要)和TLR4识别。环境pH能够引起问号钩体脂质A合成及修饰基因的表达差异,从而使其含量增加且结构发生改变。lpxC、lpxD1、lpxD2和htrB基因表达差异主要由pH的降低引起的,LA2222.LA2541和LA3996基因产物为感受pH的二元信号系统,其中LA2222和LA3996基因产物分别对lpxC和lpxD1基因的表达起调控作用,LA2541调控lpxD2和htrB基因的表达。结论：问号钩体感染细胞后其LPS合成基因尤其是脂质A合成基因有表达差异；上述基因表达差异导致问号钩体LPS含量增加和脂质A结构发生改变致使其活性发生改变；lpxC、IpxD1、lpxD2和htrB表达差异LPS含量增加和脂质A结构修饰主要由pH的降低引起的。LA2222、LA2541和LA3996基因产物为问号钩体感受pH的二元信号系统,其中LA2222和LA3996基因产物分别对lpxC和lpxD1基因的表达起调控作用,LA2541调控lpxD2和htrB基因的表达；脂质A的识别受体、诱导炎症分泌量在人和小鼠巨噬细胞中存在差异。
[Abstract]:Background and Subject : The disease of leptospirosis caused by infection of leptospirosis is one of the most popular infectious diseases in the world . The disease of leptospirosis is very similar to that of endotoxin ( also called lipopolysaccharide , lipopolysaccharide , LPS ) . The results of genome sequencing show that the disease of leptospirosis is very similar to endotoxin ( also called lipopolysaccharide , lipopolysaccharide , LPS ) .

The content and significance of LPS were studied in this paper . By studying the difference of LPS synthetic gene expression , the difference of LPS synthesis , the structure difference of lipid A in LPS , the main environmental factors affecting LPS synthesis and lipid A modification , the mechanism of lipid synthesis and modification of lipid A were investigated .

Methods : Mouse J774A . 1 and human THP - 1 mononuclear - macrophage infection model were used to determine the expression of LPS - synthesized genes in the infected cells after infection with gene chip and real - time fluorescence quantitative RT - PCR .
Limulus test was used to measure LPS activity of leptospirosis in infected cells .
The changes of LPS content in infected cells were determined by using KDO test , Purpald test , SDS - PAGE - silver staining and HPLC - MS .
The structure modification of lipid A in infected cells was determined by mass spectrometry .
The factors affecting lipid A synthesis and the expression of modified gene were determined by real - time fluorescence quantitative RT - PCR .
The regulation gene of lipid A synthesis and modification gene was determined by means of antibody blocking and gene knock - out technique .
The activity and identification of inflammatory cytokines in mononuclear - macrophages induced by lipid A in different structures were determined by means of antibody blocking and ELISA .

The results showed that there were 13 and 11 gene expression levels in the 34 LPS - synthesized genes infected with J774A - 1 and THP - 1 cells respectively .
Both lipid A - induced TNF - 伪 , IL - 1尾 and IL - 6 , but the activity of two lipid A was similar .
Both lipids A were identified by RAW264.7 cells TLR2 ( primary ) and TLR2 ( mainly ) , and the expression of the modified gene was increased by the pH of the environment . The difference of the expression of p - xC , p - xD 1 , p - xD2 and htrB genes was mainly caused by the decrease of pH . The products of LA222.LA2541 and LA3996 gene were the binary signal systems for the sensory pH .

Conclusion : The synthetic gene of LPS , especially the lipid A , is differentially expressed in the infected cells .
The difference in the expression of the above genes results in the increase of LPS content and the change of lipid A structure in question mark .
The changes of LPS and the structural modification of lipid A were mainly caused by the decrease of pH . The products of LAPA22 , LA2541 and LA3996 were the binary signal systems with the pH value of the interrogans , in which the expression of the genes LA222,LA2541 and LA3996 was regulated by the expression of p - xC and p - xD1 genes , and LA2541 regulated the expression of pxD2 and htrB genes , respectively .
The recognition receptors of lipid A , induced inflammatory secretion , differed in human and mouse macrophages .
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R377

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