本文選題：問號鉤端螺旋體 + 單核-巨噬細胞��； 參考：《浙江大學》2012年博士論文

【摘要】：背景和立題依據(jù)：由問號鉤端螺旋體(Leptospira interrogans,簡稱鉤體)感染引起的鉤體病是全世界流行的入獸共患傳染病。由于該病通過疫水迅速傳播,因而是洪澇、地震時重點監(jiān)測的傳染病之一。我國自然災(zāi)害頻發(fā),且不少地區(qū)是鉤體病流行疫區(qū)。鉤體病臨床表現(xiàn)復(fù)雜多樣,但典型癥狀和體征是黃疸和出血,其中彌漫性肺出血型患者死亡率高達50%以上。然而,問號鉤體致病機制至今不明。大量臨床病理資料揭示,鉤體病患者病變與內(nèi)毒素(又稱脂多糖,lipopolysaccharide, LPS)中毒極為相似,如各臟器廣泛、不同程度內(nèi)毒素中毒樣毛細血管及其內(nèi)皮的病變,部分肺出血型死亡患者內(nèi)臟中可見微血栓及凝血物質(zhì)水平下降等。上述資料提示,問號鉤體致病性可能與內(nèi)毒素密切相關(guān)�；蚪M測序結(jié)果表明問號鉤體賴株基因組中含有一整套的LPS合成、裝配及修飾基因,且鉤體含有LPS也已得到證明。有文獻報道問號鉤體感染豚鼠后其LPS多糖部分會發(fā)生改變,并且有報道在鉤體病葡萄膜炎患者房水中檢測出LPS,進一步表明LPS直接參與問號鉤體致病過程,且LPS會發(fā)生修飾。 研究內(nèi)容及意義：本文通過研究感染細胞后問號鉤體LPS合成基因的表達差異、LPS合成量的變化、LPS中脂質(zhì)A的結(jié)構(gòu)差異、脂質(zhì)A結(jié)構(gòu)被修飾后其活性的差異、影響LPS合成和脂質(zhì)A修飾的主要環(huán)境因素并初步探究脂質(zhì)合成和修飾基因的調(diào)控機制,以期揭示LPS在問號鉤體致病過程中的作用,并為其它革蘭陰性致病菌致病的機制研究提供參考依據(jù)。 研究方法：本文采用小鼠J774A.1和人THP-1單核-巨噬細胞感染模型,采用基因芯片和實時熒光定量RT-PCR技術(shù)測定感染細胞后問號鉤體LPS合成基因的表達差異；運用鱟試驗測定感染細胞后鉤體LPS活性變化；運用KDO試驗、Purpald試驗、SDS-PAGE-銀染色手段、HPLC-MS聯(lián)用技術(shù)測定感染細胞后LPS含量變化；采用質(zhì)譜技術(shù)測定感染細胞后脂質(zhì)A的結(jié)構(gòu)修飾；采用實時熒光定量RT-PCR技術(shù)測定影響脂質(zhì)A合成及修飾基因表達量的環(huán)境因素及調(diào)節(jié)基因；采用抗體封閉和基因敲除技術(shù)確定脂質(zhì)A合成及修飾基因的調(diào)節(jié)基因；采用抗體封閉和ELISA手段測定不同結(jié)構(gòu)脂質(zhì)A誘導(dǎo)單核-巨噬細胞分泌炎癥因子的活性及識別受體。 實驗結(jié)果：實驗結(jié)果表明,感染J774A.1和THP-1細胞后問號鉤體34個LPS合成基因中分別有13和11個基因表達水平發(fā)生變化,其中參與脂質(zhì)A合成的限速酶基因lpxB和lpxC及脂肪酸轉(zhuǎn)移酶基因lpxD2和htrB上調(diào)表達而脂肪酸轉(zhuǎn)移酶基因lpxD1下調(diào)表達。感染兩種細胞后問號鉤體脂多糖活性分別增加1.97和2.09倍,而其數(shù)量僅增加1.02和1.06倍。質(zhì)譜結(jié)果表明感染細胞后,問號鉤體由感染前含1條肉豆蔻烯酸、1條月桂烯酸、2條棕櫚酸和2條月桂酸、m/z=1921的脂質(zhì)A轉(zhuǎn)變成各有2條月桂酸、肉豆蔻二烯酸和棕櫚酸、m/z=1949的脂質(zhì)A。感染細胞后問號鉤體的脂質(zhì)A(LepLipidA-Infection)促凝活性為感染前(LepLipidA-EMJH)的3.5倍。兩種脂質(zhì)A均誘導(dǎo)人單核-巨噬細胞THP-1產(chǎn)生炎癥因子TNF-α、IL-1β及IL-8,但LepLipidA-Infection該活性較強,分別為LepLipidA-EMJH的1.95、1.95和1.69倍；LepLipidA-EMJH僅由THP-1TLR2識別,而LepLipidA-Infection由THP-1細胞TLR2(主要)和TLR4識別。兩種脂質(zhì)A均誘導(dǎo)小鼠單核-巨噬細胞RAW264.7產(chǎn)生炎癥因子TNF-α、IL-1β及IL-6,但兩種脂質(zhì)A該活性相近；兩種脂質(zhì)A均能由RAW264.7細胞TLR2(主要)和TLR4識別。環(huán)境pH能夠引起問號鉤體脂質(zhì)A合成及修飾基因的表達差異,從而使其含量增加且結(jié)構(gòu)發(fā)生改變。lpxC、lpxD1、lpxD2和htrB基因表達差異主要由pH的降低引起的,LA2222.LA2541和LA3996基因產(chǎn)物為感受pH的二元信號系統(tǒng),其中LA2222和LA3996基因產(chǎn)物分別對lpxC和lpxD1基因的表達起調(diào)控作用,LA2541調(diào)控lpxD2和htrB基因的表達。 結(jié)論：問號鉤體感染細胞后其LPS合成基因尤其是脂質(zhì)A合成基因有表達差異；上述基因表達差異導(dǎo)致問號鉤體LPS含量增加和脂質(zhì)A結(jié)構(gòu)發(fā)生改變致使其活性發(fā)生改變；lpxC、IpxD1、lpxD2和htrB表達差異LPS含量增加和脂質(zhì)A結(jié)構(gòu)修飾主要由pH的降低引起的。LA2222、LA2541和LA3996基因產(chǎn)物為問號鉤體感受pH的二元信號系統(tǒng),其中LA2222和LA3996基因產(chǎn)物分別對lpxC和lpxD1基因的表達起調(diào)控作用,LA2541調(diào)控lpxD2和htrB基因的表達；脂質(zhì)A的識別受體、誘導(dǎo)炎癥分泌量在人和小鼠巨噬細胞中存在差異。
[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 - 偽 , 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 .
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R377

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