鹦鹉热衣原体感染加重鸡H9N2亚型禽流感发病的机制研究

发布时间：2018-01-05 10:38

本文关键词：鹦鹉热衣原体感染加重鸡H9N2亚型禽流感发病的机制研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：鹦鹉热衣原体(Chlamydiapsittaci,C.psittaci)是重要的人兽共患病原之一,感染后以巨噬细胞为转运工具向全身各器官传播,欧美国家优先把C.psittaci列为潜在的生物武器。近年来,我国家禽呼吸道病呈现季节性流行,临床死亡率达到30%-40%,引发抗菌素的滥用和食品安全危机。发病鸡群中常常分离出H9N2亚型禽流感病毒、鹦鹉热衣原体和大肠杆菌。康复鸡群中鹦鹉热衣原体、偏肺病毒和鼻气管鸟疫杆菌呈现较高的血清阳性率。因此,探索鹦鹉热衣原体、H9N2禽流感H9N2在家禽持续性呼吸道中的作用机制可以为防控家禽呼吸道疾病提供理论基础。衣原体多型外膜蛋白家族(Polymorphic membrane proteins,Pmps)存在于所有衣原体种属中,并已证明沙眼衣原体Pmps蛋白家族中的PmpD是毒力因子,但是禽鹦鹉热衣原体PmpD是否具有类似的作用,是否抑制巨噬细胞功能并在衣原体免疫逃逸中发挥作用?因此,本研究提出以下科学假说:(1)C.psittaci通过分泌PmpD-N直接抑制巨噬细胞功能而降低巨噬细胞杀灭C.psittaci和H9N2的效率;(2)分泌的PmpD-N通过与Toll-like受体结合激活TLRs/MyD88/NF-KB信号通路,诱导Th1/Th2的失衡而下调巨噬细胞的先天性免疫功能,衣原体实现免疫逃避而扩散感染。为了验证以上科学假说,本课题开展以下研究工作:(1)C.psittaci与H9N2单独、混合感染对SPF鸡致病力的影响;(2)体外试验验证C.psittaci与H9N2单独、混合感染对巨噬细胞HD11的影响以及TLRs相关信号通路的表达特点;(3)C.psittaci分泌的PmpD-N作用巨噬细胞HD11后相关TLRs/MyD88/NF-KB信号通路表达特点以及Th1/Th2型细胞因子表达特点。同时,以siRNA干扰和信号通路的拮抗剂验证以上结论。试验1:C.psttaci与H9N2单独、混合感染对SPF鸡致病性的影响。以不同致病力C.psittaci感染SPF鸡,试验发现高致病力C.psittaci感染后鸡群新城疫抗体水平显著下降(P0.01)。C.paittaci和H9N2同时感染、先感染Cp.ittaci后感染H9N2,鸡群体重、免疫器官指数和细胞因子表达水平均显著降低,死亡率和免疫器官损伤加重(P0.01),靶组织中H9N2病毒载量显著性增高(P0.01)。这种感染模型不但复制了家禽严重的呼吸道病和诱导较高的死亡率,而且发现高致病力的C.psittaci可以造成体液免疫和细胞免疫下调,导致机体免疫功能受到抑制,从而有助于H9N2病毒的继发感染。试验2:C.psittaci与H9N2单独、混合感染对巨噬细胞HD11功能的影响。以鸡巨噬细胞HD11细胞系为研究对象,采用不同顺序感染C.psittaci或H9N2。结果显示,初次感染C.psittaci会显著提高H9N2病毒感染细胞的死亡,降低HD1]细胞的吞噬能力和iNOS活性(P0.01)。同时,C.psittact感染会显著下调H9N2诱导的巨噬细胞促炎性细胞因子的表达(P0.01)。试验3:鹦鹉热衣原体PmpD-N蛋白对HD11巨噬细胞功能的影响。首先构建了pmpD-N基因的真核表达质粒pEGFP-N1-pmpD-N,将重组质粒导入HD11细胞中,RT-PCR方法证明转染重组质粒pEGFP-N1-pmpD-N的HD11细胞能够编码PmpD-N蛋白的mRNA;Western Blot证实pmpD-N基因在HD11细胞中得到表达。其次,分别以PmpD-N和重组真核表达质粒pEGFP-N1-pmpD-N与HD11细胞作用,以LPS为阳性对照,利用荧光抗体染色技术测定衣原体的感染量,以荧光微球法测定HD11细胞吞噬功能,采用Griess法检测细胞培养液上清中NO的含量。结果显示PmpD-N与HD11作用后显著降低衣原体的感染和吞噬能力(P0.01),并不提高NO的分泌水平(P0.05),显著上调细胞因子IL-6、IL-10以及模式识别受体TLR2、TLR4和TLR5的mRNA表达水平(P0.01),其中以TLR2上调水平最为显著,MyD88和转录因子NF-κB的mRNA表达水平也有不同程度的提高,这提示PmpD-N激活了 TLR2/MyD88信号通路并诱导Th2型细胞免疫应答。试验4:TLR2/MyD88/NF-κB信号通路参与PmpD-N下调HD11功能的验证。分别以PmpD-N、pEGFP-N1-pmpD-N 与 HD11 细胞作用,以 LPS 为阳性对照。Western Blot 方法检测 TLRs、MyD88的表达特点,以Confocal技术检测NF-κBp65激活向细胞核迁徙特点,以EMSA检测p65与特异性DNA靶向结合情况,分析NF-κB进入细胞核后启动目的基因转录的情况。利用RNA干扰和NF-κB抑制剂PDTC,验证信号通路分子被干扰或阻断后PmpD-N刺激对HD11功能影响的变化。结果显示TLR2、MyD88表达显著上调(P0.01),p65向细胞核内迁移并与相应的启动子基因结合后启动基因的转录,证实激活了 TLR2/MyD88/NF-κB信号通路。采用RNA干扰技术干扰TLR2、MyD88和使用抑制剂阻断NF-κB后,由PmpD-N刺激HD11细胞产生的IL-6、IL-10水平显著下调(P0.01),PmpD-N诱导HD11细胞后Th1/Th2平衡出现新的状态。综上所述,本研究整体动物试验不但为家禽呼吸道发病提供了良好的发病模型,且首次证实,鹦鹉热衣原体和PmpD-N通过直接下调巨噬细胞功能导致鸡免疫功能下降,从而更有利于禽流感H9N2的发病。体外试验和验证试验显示PmpD-N下调巨噬细胞功能还可以依赖于间接途径:通过调控TLR2/MyD88/NF-κB信号通路和诱导Th1/T2失衡,下调HD11巨噬细胞功能,从而有利于衣原体在HD11细胞中的存活实现免疫逃逸。
[Abstract]:Chlamydia psittaci (Chlamydiapsittaci, C.psittaci) is one of the important zoonotic pathogens, infected macrophages as transport spread to all organs of the body, the European and American countries preferred to put C.psittaci as potential biological weapons. In recent years, poultry respiratory disease in China presents seasonal epidemic, clinical mortality rate reached 30%-40%, and the abuse of food safety crisis antibiotics. Chickens are often isolated from H9N2 subtype avian influenza virus, Chlamydia psittaci and Escherichia coli. Chlamydia psittaci in chickens in rehabilitation, the positive rate of serum human metapneumovirus and ornithosis coli showed a higher nasal trachea. Therefore, exploration of Chlamydia psittaci, avian influenza H9N2 H9N2 can provide a theoretical basis for the prevention and control of respiratory diseases of poultry in the mechanism of persistent respiratory tract in poultry. Chlamydia type outer membrane protein family (Polymorphic membrane, proteins, Pmps) Chlamydia exists in all species, and it is proved that the Chlamydia trachomatis Pmps protein family in PmpD is a virulence factor, but avian Chlamydia psittaci PmpD has similar effect on whether inhibition of macrophage function and play a role in the immune escape of chlamydia? Therefore, this study proposed the following hypothesis: (1) C.psittaci by secreting PmpD-N directly inhibition of macrophage function and reduce the efficiency of macrophage killing C.psittaci and H9N2; (2) the secretion of PmpD-N by binding to Toll-like receptor TLRs/MyD88/NF-KB signaling pathway activated innate immune function induced by Th1/ Th2 imbalance while downregulation of macrophages, immune escape and Chlamydia infection diffusion. In order to verify the above hypothesis, this paper carried out the following research work: (1) C.psittaci and H9N2 separately, the mixed infection effect on SPF chicken pathogenicity test in vitro; (2) C.psittaci With H9N2 alone, the mixed infection effect on macrophage HD11 and expression of TLRs signaling pathway; (3) related to TLRs/MyD88/NF-KB signaling pathway expression and characteristics of Th1/Th2 type cytokine macrophage HD11 PmpD-N effect on C.psittaci secretion. At the same time, the antagonism of siRNA interference and signal transduction agent to verify the above conclusions. The test of 1:C.psttaci and H9N2 alone, the mixed infection of chicken pathogenic effects on SPF. The pathogenicity of different C.psittaci infected SPF chickens, found high pathogenic C.psittaci infected chicken Newcastle disease antibody level was significantly lower (P0.01) Co infection with.C.paittaci and H9N2, the first Cp.ittaci infection after H9N2 infection in chickens, body weight, immune organ index and expression level cytokines significantly decreased mortality and immune organ injury (P0.01), H9N2 viral load was significantly higher in target tissues (P0.01). This kind of feeling Dye model not only copied poultry respiratory disease serious and induced higher mortality, and found that the highly pathogenic C.psittaci can cause humoral and cellular immune down-regulation, cause immune function was inhibited, which contributes to the secondary infection of H9N2 virus. 2: C.psittaci test with H9N2 alone, the mixed infection effect on macrophage function of HD11 to chicken macrophage cell line HD11 as the research object, using C.psittaci or H9N2. showed different sequence of infection, primary C.psittaci infection can significantly increase cell death of H9N2 virus infection, lower HD1] cell phagocytosis and the activity of iNOS (P0.01). At the same time, the expression of C.psittact infection can significantly reduce H9N2 induced macrophage inflammatory cells the impact factor (P0.01). 3: test of Chlamydia psittaci PmpD-N protein on HD11 macrophage. Firstly pmpD-N gene really The eukaryotic expression plasmid pEGFP-N1-pmpD-N, the recombinant plasmid was transfected into HD11 cells, RT-PCR assay showed that the recombinant plasmid pEGFP-N1-pmpD-N HD11 encoding PmpD-N protein of mRNA cells; Western Blot confirmed that the pmpD-N gene expressed in HD11 cells. Secondly, using PmpD-N and the recombinant eukaryotic expression plasmid pEGFP-N1-pmpD-N and HD11 cells, LPS was used as positive control the amount of infection, staining technique for determination of Chlamydia by fluorescent antibody, determination of phagocytosis of HD11 cells by fluorescent microsphere method, detection of cell culture supernatant NO content by Griess method. The results showed that PmpD-N and HD11 decreased significantly after Chlamydia infection and phagocytosis (P0.01), does not increase the secretion level of NO (P0.05) a significant increase in cell factor, IL-6, IL-10 and pattern recognition receptor TLR2, TLR4 and TLR5 expression level of mRNA (P0.01), and the increase of TLR2 level is the most significant The MyD88 and NF- transcription factor kappa B mRNA expression levels also have different degrees of increase, suggesting that PmpD-N activation of the TLR2/MyD88 signaling pathway and induce Th2 type immune response. Test of 4:TLR2/MyD88/NF- kappa B signaling pathway is involved in the downregulation of HD11. Verify the PmpD-N were PmpD-N, pEGFP-N1-pmpD-N and HD11 cells, with LPS positive.Western control method for the detection of Blot TLRs, expression of MyD88, Confocal technology to detect NF- kappa Bp65 activation to nuclear migration characteristics, detected by EMSA p65 and specific DNA targeting, analysis of NF- kappa B into the cell nucleus after gene transcription. Using RNA interference and NF- kappa B inhibitor PDTC, change effect of PmpD-N stimulation on the function of HD11 signaling pathway molecules are verified. The results showed that interference or blocking TLR2, MyD88 expression was significantly up-regulated (P0.01, p65) to the nucleus and the corresponding migration The gene promoter with gene transcription, confirmed the activation of TLR2/MyD88/NF- B signaling pathway. The interference of RNA interference of TLR2, MyD88 and NF- using the inhibitor kappa B, produced by PmpD-N stimulated HD11 cell IL-6, IL-10 levels were significantly reduced (P0.01), PmpD-N HD11 cells induced by Th1/Th2 after the emergence of new balance state. To sum up, the study of animal experiment for poultry respiratory disease not only provides a good model of the disease, and for the first time that c.psittaci and PmpD-N through direct down-regulation of macrophage function in chicken immune function decline, and thus more conducive to the pathogenesis of avian influenza H9N2. In vitro experiments showed that PmpD-N down-regulation of macrophage function can also depend on indirect way: by regulation of TLR2/MyD88/NF- B signaling pathway and Th1/T2 induced down-regulation of macrophage function imbalance, HD11, which is beneficial to the original clothes The survival of the body in HD11 cells is immune escape.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.31

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