HBsAg干扰巨噬样细胞TLR信号通路的初步研究

发布时间：2018-01-15 19:02

  本文关键词：HBsAg干扰巨噬样细胞TLR信号通路的初步研究　出处：《复旦大学》2006年硕士论文　论文类型：学位论文

  更多相关文章： 乙肝表面抗原 Toll样受体 巨噬样细胞 THP-1 SOCS1

【摘要】： 乙型肝炎病毒(hepatitis B virus,HBV)是一种有不完全环状双链DNA的胞膜病毒,感染人体后引起不同的临床表现,有一过性的急性肝炎、持续性慢性肝炎,甚至致死性的暴发性肝炎。现在一般认为免疫系统介导的宿主与病毒的相互作用决定着HBV感染后病毒的清除和肝炎病变的进程。多个研究显示,机体对HBV特异性细胞免疫反应的活力、多样性及效应功能是HBV感染后转归的关键,因此,机体细胞免疫反应功能障碍被认为是HBV持续性感染的重要原因。目前造成HBV持续性感染患者T细胞反应障碍的原因还不清楚,但启动特异性T细胞反应的抗原呈递功能受阻一定在其中发挥作用。Toll样受体(Toll likereceptors,TLRs)家族属于构型识别受体(pattern recognition receptors,PRRs)成员,介导识别病原体保守的分子结构,在天然免疫系统中起着非常重要的作用。近年研究发现,TLR信号通路调节的抗原呈递细胞活化是启动正常T细胞免疫反应的前提,而一些病毒的蛋白通过与TLRs及其下游的信号分子相互作用干扰TLR信号通路的活化,这提示病毒可以通过影响TLR信号通路的活化来调节免疫系统的抗原呈递。HBV的病毒蛋白是否也能利用这种免疫调节机制则有待证明。 为此本研究选用THP-1分化的巨噬样细胞作为研究对象,观察HBV感染后分泌产生最多的胞膜蛋白—乙肝表面抗原(hepatitis B surface antigen,HBsAg)对TLR信号通路活化的影响。首先通过Real-time PCR的方法检测了THP-1和THP-1在PMA刺激下诱导分化的巨噬样细胞的TLR表达谱。结果显示,THP-1分化为巨噬样细胞后,多数TLRs的表达有明显的增加。且在巨噬样细胞中,TLR1、TLR2、TLR4和TLR6的表达明显高于其他TLRs。为在TLR信号通路充分活化的情况下研究HBsAg的调节效应,选择激活巨噬样细胞上高表达的TLRs信号通路作进一步研究。TLR1/2的配体pam3csk4和TLR4的配体LPS刺激细胞后,通过Real-time PCR和ELISA分别从蛋白水平和mRNA水平的检测IL-10和IL-12表达,结果显示HBsAg的胞外处理能以剂量依赖的方式干扰pam3csk4和LPS诱导的IL-10和IL-12的产生,提示HBsAg能干扰巨噬样细胞TLR信号通路活化。为了解HBsAg对TLR下游信号通路的影响,利用免疫荧光分析了LPS和pam3csk4诱导的NF-κB p65入核情况。结果发现,HBsAg的存在明显干扰pam3csk4和LPS诱导的NF-κB p65入核,Western blotting检测结果同样显示,HBsAg能抑制诱导的pam3csk4和LPS诱导的IκB-α降解,这提示HBsAg对TLRs下游的NF-κB通路有干扰效应。进一步用Western blotting检测磷酸化EEK蛋白的表达,结果显示HBsAg阻止了pam3csk4和LPS诱导的ERK蛋白磷酸化,说明HBsAg对TLRs下游的ERK通路也有影响。上述结果再次证明HBsAg对TLR信号通路活化的抑制作用。为进一步了解HBsAg干扰TLR信号通路的机制,Real-time PCR检测了HBsAg的处理对巨噬样细胞TLRs表达的影响。结果显示,HBsAg并不依赖下调TLRs的表达来干扰TLR信号通路活化,甚至HBsAg能上调TLR4的表达。除了干扰TLRs的膜外识别,细胞内的调节因子也能影响TLR信号通路的活化。现已发现多种调节因子可影响TLR信号的传导,包括MyD88s、IRAK3、Tollip、IRF家族的IRF4与IRF5及SOCS家族的SOCS1和SOCS3等。通过Real-time PCR分析HBsAg胞外处理对这些调节因子的表达影响,结果发现HBsAg能上调SOCS1的表达,提示HBsAg可能通过上调SOCS1来干扰巨噬样细胞上TLR通路的活化。 综上所述,我们的结果提示HBV感染后产生的高载量HBsAg,除了可能引起T细胞耗竭直接影响获得性免疫反应外,还可能影响固有免疫反应,通过干扰TLR信号通路的活化来影响抗原呈递,阻止HBV特异型T细胞免疫反应的建立。本研究的进一步开展将有助于了解HBV持续性感染者T细胞反应低下的原因,为探讨慢性HBV感染的致病机制及研制新型抗HBV药物提供新的理论依据。
[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.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【共引文献】

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6 齐，

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