本文關(guān)鍵詞：HBsAg干擾巨噬樣細(xì)胞TLR信號通路的初步研究　出處：《復(fù)旦大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乙肝表面抗原 Toll樣受體 巨噬樣細(xì)胞 THP-1 SOCS1

【摘要】： 乙型肝炎病毒(hepatitis B virus,HBV)是一種有不完全環(huán)狀雙鏈DNA的胞膜病毒,感染人體后引起不同的臨床表現(xiàn),有一過性的急性肝炎、持續(xù)性慢性肝炎,甚至致死性的暴發(fā)性肝炎�，F(xiàn)在一般認(rèn)為免疫系統(tǒng)介導(dǎo)的宿主與病毒的相互作用決定著HBV感染后病毒的清除和肝炎病變的進(jìn)程。多個研究顯示,機體對HBV特異性細(xì)胞免疫反應(yīng)的活力、多樣性及效應(yīng)功能是HBV感染后轉(zhuǎn)歸的關(guān)鍵,因此,機體細(xì)胞免疫反應(yīng)功能障礙被認(rèn)為是HBV持續(xù)性感染的重要原因。目前造成HBV持續(xù)性感染患者T細(xì)胞反應(yīng)障礙的原因還不清楚,但啟動特異性T細(xì)胞反應(yīng)的抗原呈遞功能受阻一定在其中發(fā)揮作用。Toll樣受體(Toll likereceptors,TLRs)家族屬于構(gòu)型識別受體(pattern recognition receptors,PRRs)成員,介導(dǎo)識別病原體保守的分子結(jié)構(gòu),在天然免疫系統(tǒng)中起著非常重要的作用。近年研究發(fā)現(xiàn),TLR信號通路調(diào)節(jié)的抗原呈遞細(xì)胞活化是啟動正常T細(xì)胞免疫反應(yīng)的前提,而一些病毒的蛋白通過與TLRs及其下游的信號分子相互作用干擾TLR信號通路的活化,這提示病毒可以通過影響TLR信號通路的活化來調(diào)節(jié)免疫系統(tǒng)的抗原呈遞。HBV的病毒蛋白是否也能利用這種免疫調(diào)節(jié)機制則有待證明。 為此本研究選用THP-1分化的巨噬樣細(xì)胞作為研究對象,觀察HBV感染后分泌產(chǎn)生最多的胞膜蛋白—乙肝表面抗原(hepatitis B surface antigen,HBsAg)對TLR信號通路活化的影響。首先通過Real-time PCR的方法檢測了THP-1和THP-1在PMA刺激下誘導(dǎo)分化的巨噬樣細(xì)胞的TLR表達(dá)譜。結(jié)果顯示,THP-1分化為巨噬樣細(xì)胞后,多數(shù)TLRs的表達(dá)有明顯的增加。且在巨噬樣細(xì)胞中,TLR1、TLR2、TLR4和TLR6的表達(dá)明顯高于其他TLRs。為在TLR信號通路充分活化的情況下研究HBsAg的調(diào)節(jié)效應(yīng),選擇激活巨噬樣細(xì)胞上高表達(dá)的TLRs信號通路作進(jìn)一步研究。TLR1/2的配體pam3csk4和TLR4的配體LPS刺激細(xì)胞后,通過Real-time PCR和ELISA分別從蛋白水平和mRNA水平的檢測IL-10和IL-12表達(dá),結(jié)果顯示HBsAg的胞外處理能以劑量依賴的方式干擾pam3csk4和LPS誘導(dǎo)的IL-10和IL-12的產(chǎn)生,提示HBsAg能干擾巨噬樣細(xì)胞TLR信號通路活化。為了解HBsAg對TLR下游信號通路的影響,利用免疫熒光分析了LPS和pam3csk4誘導(dǎo)的NF-κB p65入核情況。結(jié)果發(fā)現(xiàn),HBsAg的存在明顯干擾pam3csk4和LPS誘導(dǎo)的NF-κB p65入核,Western blotting檢測結(jié)果同樣顯示,HBsAg能抑制誘導(dǎo)的pam3csk4和LPS誘導(dǎo)的IκB-α降解,這提示HBsAg對TLRs下游的NF-κB通路有干擾效應(yīng)。進(jìn)一步用Western blotting檢測磷酸化EEK蛋白的表達(dá),結(jié)果顯示HBsAg阻止了pam3csk4和LPS誘導(dǎo)的ERK蛋白磷酸化,說明HBsAg對TLRs下游的ERK通路也有影響。上述結(jié)果再次證明HBsAg對TLR信號通路活化的抑制作用。為進(jìn)一步了解HBsAg干擾TLR信號通路的機制,Real-time PCR檢測了HBsAg的處理對巨噬樣細(xì)胞TLRs表達(dá)的影響。結(jié)果顯示,HBsAg并不依賴下調(diào)TLRs的表達(dá)來干擾TLR信號通路活化,甚至HBsAg能上調(diào)TLR4的表達(dá)。除了干擾TLRs的膜外識別,細(xì)胞內(nèi)的調(diào)節(jié)因子也能影響TLR信號通路的活化�，F(xiàn)已發(fā)現(xiàn)多種調(diào)節(jié)因子可影響TLR信號的傳導(dǎo),包括MyD88s、IRAK3、Tollip、IRF家族的IRF4與IRF5及SOCS家族的SOCS1和SOCS3等。通過Real-time PCR分析HBsAg胞外處理對這些調(diào)節(jié)因子的表達(dá)影響,結(jié)果發(fā)現(xiàn)HBsAg能上調(diào)SOCS1的表達(dá),提示HBsAg可能通過上調(diào)SOCS1來干擾巨噬樣細(xì)胞上TLR通路的活化。 綜上所述,我們的結(jié)果提示HBV感染后產(chǎn)生的高載量HBsAg,除了可能引起T細(xì)胞耗竭直接影響獲得性免疫反應(yīng)外,還可能影響固有免疫反應(yīng),通過干擾TLR信號通路的活化來影響抗原呈遞,阻止HBV特異型T細(xì)胞免疫反應(yīng)的建立。本研究的進(jìn)一步開展將有助于了解HBV持續(xù)性感染者T細(xì)胞反應(yīng)低下的原因,為探討慢性HBV感染的致病機制及研制新型抗HBV藥物提供新的理論依據(jù)。
[Abstract]:Hepatitis B virus (hepatitis B, virus, HBV) is a membrane virus a partially double stranded DNA, human infection caused by different clinical manifestations, had acute hepatitis, chronic persistent hepatitis, even fatal fulminant hepatitis. It is now generally accepted that the clearance and interaction of hepatitis disease free host and virus mediated immune system determines HBV infection virus process. Many studies show that the body of HBV specific cellular immune response activity, diversity and function is the key to the outcome of HBV infection after the immune response dysfunction is considered to be an important cause of persistent HBV at present, the cause of infection. HBV cell responses in patients with persistent T infection disorder is not clear, but the start of the antigen presentation function of specific T cell responses blocked must play the role of.Toll like receptors in the Toll (liker Eceptors, TLRs) belongs to a family of pattern recognition receptors (pattern, recognition receptors, PRRs) members, mediated by the molecular structure of conserved pathogen recognition, plays a very important role in the innate immune system. Recent studies have found that antigen-presenting cells regulated by TLR signaling pathway activation is the premise to start normal T cell immune reaction, and some the virus protein by TLRs and its downstream signal molecules and the interaction of activation of TLR signaling, whether the virus protein antigen presenting.HBV which indicated that the virus can regulate the immune system by influencing the activation of TLR signaling pathway can also use this kind of immune regulation mechanism has yet to be proved.
This research used the THP-1 differentiation of macrophage like cells as the research object, observe the HBV after infection of hepatitis B surface antigen secreted protein membrane (hepatitis B surface antigen up, HBsAg) on the activation of the TLR pathway. Firstly, method of Real-time detection of PCR THP-1 and THP-1 under PMA stimulation induced differentiation macrophage like cell TLR expression. The results showed that THP-1 differentiate into macrophage like cells, the expression of the majority of TLRs has increased significantly. And in macrophage like cells, TLR1, TLR2, TLR4 and TLR6 expression regulation effect was significantly higher than that of other TLRs. for full activation of TLR signaling pathway in the case study on the selection of HBsAg, TLRs signaling pathway activated macrophage like cells with high expression for the further study of.TLR1/2 and TLR4 ligand pam3csk4 ligand LPS cells stimulated by Real-time, PCR and ELISA respectively from the protein level and mR The expression level of NA in the detection of IL-10 and IL-12, results showed that the extracellular HBsAg treatment pam3csk4 and LPS interference in a dose dependent induction of IL-10 and IL-12, suggesting that HBsAg can interfere with macrophage like cell activation of TLR signaling pathway. In order to understand the HBsAg of the TLR signaling pathways influence analysis of LPS and pam3csk4 induction of NF- kappa B nuclear translocation of p65 by immunofluorescence. The results showed that HBsAg has obvious interference pam3csk4 and LPS induced NF- kappa B nuclear translocation of p65, Western, blotting test results also showed that HBsAg could inhibit pam3csk4 induced and LPS induced I kappa B- alpha degradation, suggesting that the HBsAg of TLRs downstream NF- B pathway interference effect. Further detection of blotting expression by Western phosphorylation of EEK protein, the results showed that HBsAg prevented ERK phosphorylation induced by LPS and pam3csk4, that also has the effect of HBsAg on the downstream of the TLRs ERK pathway. The results proved once again that the inhibitory effect of HBsAg on activation of TLR signaling pathway. To further understand the mechanism of HBsAg interference of TLR signaling pathway, Real-time PCR tested the effect of HBsAg treatment on the expression of macrophage like cell TLRs. The results showed that HBsAg is not dependent on expression of TLRs to interfere with the activation of the TLR pathway, and HBsAg can up regulate the expression of TLR4 the interference of TLRs. In addition to membrane recognition, regulator cells can also influence the activation of TLR signaling pathway. It has been found that many factors can affect the regulation of TLR signal transduction, including MyD88s, IRAK3, Tollip, IRF4 and IRF5 and the SOCS IRF family of SOCS1 and SOCS3 by Real-time PCR analysis of HBsAg cell. Treatment effects on these factors regulating the expression of the results showed that HBsAg could increase the expression of SOCS1, suggesting that HBsAg may regulate SOCS1 activation of the TLR pathway to interfere with macrophage like cells.
To sum up, the high load HBsAg our results suggest that HBV infection, but may cause depletion of T cells directly affect the acquired immune response, may also affect the innate immune response activated by interfering with TLR signaling pathway to influence antigen presentation, a stop HBV specific type T cell immune response. This study further carry out will be helpful to understand the causes of persistent HBV infected T cell reaction is low, in order to explore the pathogenic mechanism of chronic HBV infection and provide a new theoretical basis for the development of new anti HBV drugs.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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