發(fā)布時間：2018-01-05 10:38

  本文關(guān)鍵詞：鸚鵡熱衣原體感染加重雞H9N2亞型禽流感發(fā)病的機制研究　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鸚鵡熱衣原體 禽流感病毒H9N2亞型 PmpD蛋白 雞巨噬細胞

【摘要】：鸚鵡熱衣原體(Chlamydiapsittaci,C.psittaci)是重要的人獸共患病原之一,感染后以巨噬細胞為轉(zhuǎn)運工具向全身各器官傳播,歐美國家優(yōu)先把C.psittaci列為潛在的生物武器。近年來,我國家禽呼吸道病呈現(xiàn)季節(jié)性流行,臨床死亡率達到30%-40%,引發(fā)抗菌素的濫用和食品安全危機。發(fā)病雞群中常常分離出H9N2亞型禽流感病毒、鸚鵡熱衣原體和大腸桿菌。康復(fù)雞群中鸚鵡熱衣原體、偏肺病毒和鼻氣管鳥疫桿菌呈現(xiàn)較高的血清陽性率。因此,探索鸚鵡熱衣原體、H9N2禽流感H9N2在家禽持續(xù)性呼吸道中的作用機制可以為防控家禽呼吸道疾病提供理論基礎(chǔ)。衣原體多型外膜蛋白家族(Polymorphic membrane proteins,Pmps)存在于所有衣原體種屬中,并已證明沙眼衣原體Pmps蛋白家族中的PmpD是毒力因子,但是禽鸚鵡熱衣原體PmpD是否具有類似的作用,是否抑制巨噬細胞功能并在衣原體免疫逃逸中發(fā)揮作用?因此,本研究提出以下科學(xué)假說:(1)C.psittaci通過分泌PmpD-N直接抑制巨噬細胞功能而降低巨噬細胞殺滅C.psittaci和H9N2的效率;(2)分泌的PmpD-N通過與Toll-like受體結(jié)合激活TLRs/MyD88/NF-KB信號通路,誘導(dǎo)Th1/Th2的失衡而下調(diào)巨噬細胞的先天性免疫功能,衣原體實現(xiàn)免疫逃避而擴散感染。為了驗證以上科學(xué)假說,本課題開展以下研究工作:(1)C.psittaci與H9N2單獨、混合感染對SPF雞致病力的影響;(2)體外試驗驗證C.psittaci與H9N2單獨、混合感染對巨噬細胞HD11的影響以及TLRs相關(guān)信號通路的表達特點;(3)C.psittaci分泌的PmpD-N作用巨噬細胞HD11后相關(guān)TLRs/MyD88/NF-KB信號通路表達特點以及Th1/Th2型細胞因子表達特點。同時,以siRNA干擾和信號通路的拮抗劑驗證以上結(jié)論。試驗1:C.psttaci與H9N2單獨、混合感染對SPF雞致病性的影響。以不同致病力C.psittaci感染SPF雞,試驗發(fā)現(xiàn)高致病力C.psittaci感染后雞群新城疫抗體水平顯著下降(P0.01)。C.paittaci和H9N2同時感染、先感染Cp.ittaci后感染H9N2,雞群體重、免疫器官指數(shù)和細胞因子表達水平均顯著降低,死亡率和免疫器官損傷加重(P0.01),靶組織中H9N2病毒載量顯著性增高(P0.01)。這種感染模型不但復(fù)制了家禽嚴(yán)重的呼吸道病和誘導(dǎo)較高的死亡率,而且發(fā)現(xiàn)高致病力的C.psittaci可以造成體液免疫和細胞免疫下調(diào),導(dǎo)致機體免疫功能受到抑制,從而有助于H9N2病毒的繼發(fā)感染。試驗2:C.psittaci與H9N2單獨、混合感染對巨噬細胞HD11功能的影響。以雞巨噬細胞HD11細胞系為研究對象,采用不同順序感染C.psittaci或H9N2。結(jié)果顯示,初次感染C.psittaci會顯著提高H9N2病毒感染細胞的死亡,降低HD1]細胞的吞噬能力和iNOS活性(P0.01)。同時,C.psittact感染會顯著下調(diào)H9N2誘導(dǎo)的巨噬細胞促炎性細胞因子的表達(P0.01)。試驗3:鸚鵡熱衣原體PmpD-N蛋白對HD11巨噬細胞功能的影響。首先構(gòu)建了pmpD-N基因的真核表達質(zhì)粒pEGFP-N1-pmpD-N,將重組質(zhì)粒導(dǎo)入HD11細胞中,RT-PCR方法證明轉(zhuǎn)染重組質(zhì)粒pEGFP-N1-pmpD-N的HD11細胞能夠編碼PmpD-N蛋白的mRNA;Western Blot證實pmpD-N基因在HD11細胞中得到表達。其次,分別以PmpD-N和重組真核表達質(zhì)粒pEGFP-N1-pmpD-N與HD11細胞作用,以LPS為陽性對照,利用熒光抗體染色技術(shù)測定衣原體的感染量,以熒光微球法測定HD11細胞吞噬功能,采用Griess法檢測細胞培養(yǎng)液上清中NO的含量。結(jié)果顯示PmpD-N與HD11作用后顯著降低衣原體的感染和吞噬能力(P0.01),并不提高NO的分泌水平(P0.05),顯著上調(diào)細胞因子IL-6、IL-10以及模式識別受體TLR2、TLR4和TLR5的mRNA表達水平(P0.01),其中以TLR2上調(diào)水平最為顯著,MyD88和轉(zhuǎn)錄因子NF-κB的mRNA表達水平也有不同程度的提高,這提示PmpD-N激活了 TLR2/MyD88信號通路并誘導(dǎo)Th2型細胞免疫應(yīng)答。試驗4:TLR2/MyD88/NF-κB信號通路參與PmpD-N下調(diào)HD11功能的驗證。分別以PmpD-N、pEGFP-N1-pmpD-N 與 HD11 細胞作用,以 LPS 為陽性對照。Western Blot 方法檢測 TLRs、MyD88的表達特點,以Confocal技術(shù)檢測NF-κBp65激活向細胞核遷徙特點,以EMSA檢測p65與特異性DNA靶向結(jié)合情況,分析NF-κB進入細胞核后啟動目的基因轉(zhuǎn)錄的情況。利用RNA干擾和NF-κB抑制劑PDTC,驗證信號通路分子被干擾或阻斷后PmpD-N刺激對HD11功能影響的變化。結(jié)果顯示TLR2、MyD88表達顯著上調(diào)(P0.01),p65向細胞核內(nèi)遷移并與相應(yīng)的啟動子基因結(jié)合后啟動基因的轉(zhuǎn)錄,證實激活了 TLR2/MyD88/NF-κB信號通路。采用RNA干擾技術(shù)干擾TLR2、MyD88和使用抑制劑阻斷NF-κB后,由PmpD-N刺激HD11細胞產(chǎn)生的IL-6、IL-10水平顯著下調(diào)(P0.01),PmpD-N誘導(dǎo)HD11細胞后Th1/Th2平衡出現(xiàn)新的狀態(tài)。綜上所述,本研究整體動物試驗不但為家禽呼吸道發(fā)病提供了良好的發(fā)病模型,且首次證實,鸚鵡熱衣原體和PmpD-N通過直接下調(diào)巨噬細胞功能導(dǎo)致雞免疫功能下降,從而更有利于禽流感H9N2的發(fā)病。體外試驗和驗證試驗顯示PmpD-N下調(diào)巨噬細胞功能還可以依賴于間接途徑:通過調(diào)控TLR2/MyD88/NF-κB信號通路和誘導(dǎo)Th1/T2失衡,下調(diào)HD11巨噬細胞功能,從而有利于衣原體在HD11細胞中的存活實現(xiàn)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.

【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S858.31

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