【摘要】：結核病(Tuberculosis, TB)是主要由結核分枝桿菌(Mycobacterium tuberculosis, MTB)引起的一類以呼吸系統(tǒng)感染為主的慢性人獸共患傳染病。TB是一種古老的疾病,在5000余年前的埃及就發(fā)現了該病的存在,自19世紀末發(fā)現TB的致病原以來,人類對它們的研究已經持續(xù)了一百多年。然而,TB仍是目前世界上最重要的細菌性傳染病,全球約I/3人口已被TB病原菌感染,每年有約800萬TB新增病例和130萬死亡病例,是人類傳染病中最大的單因素致死疾病和全球主要疾病負擔之一MTB作為胞內菌,其引起的慢性傳染多數處于潛伏感染狀態(tài),機體產生的抗菌免疫應答決定疾病的發(fā)生和發(fā)展。樹突狀細胞(Dendritic cells, DCs)作為體內功能最強的抗原提呈細胞,通過提呈抗原觸發(fā)初始T細胞免疫應答并分泌IL-12等細胞因子的方式在機體抗TB免疫應答中發(fā)揮關鍵作用,是聯系天然免疫和獲得性免疫應答的橋梁。然而,DCs是由許多表型和功能不同的亞型組成的高度異質性群體,穩(wěn)態(tài)時小鼠脾臟DCs主要包括漿細胞DC(plasmacytoid DCs, pDCs)和經典DC (conventional DC, cDC),后者又包括CD8+ cDC和CD8+ cDC等亞型。對不同DC亞型在抗TB免疫應答中的具體免疫功能的深入研究將極大促進對機體抗TB應答過程的了解。然而,由于DCs數量稀少以及亞型分選和制備困難等原因,目前對不同DC亞型在機體抗分枝桿菌免疫應答中的作用還所知甚少。本研究在體內和體外條件下對小鼠不同亞型樹突狀細胞在BCG感染過程中的免疫應答特性進行了分析,并比較了不同DC亞型在BCG體外感染早期的基因表達譜差異,以期增加對不同DC亞型在機體抗分枝桿菌免疫應答中的作用的了解。1、小鼠骨髓源DC亞型的體外誘導、鑒定及其生物學特性分析通過小鼠Flt3L蛋白體外誘導骨髓造血干細胞制備DCs的cDC和pDC亞型,觀察誘導過程中細胞形態(tài)學變化,分析其特征性分子標志的表達,并對其活化前后共刺激分子的表達、細胞因子表達譜以及誘導T細胞增殖的能力等功能特征進行分析。流式細胞術(FCM)分析顯示體外培養(yǎng)9 d后CD11c的陽性率可達60%以上,經LPS活化后在光學顯微鏡下可觀察到典型的樹突狀形態(tài)。誘導后獲得了具備CD11c+CD45RA-和CD11c+CD45RA+表型特征的cDC和pDC亞型,且cDC可進一步分為CD24highCD11blow和CD24lowCDbhigh兩個亞型,同時,pDC也特異性表達mPDCA-1分子標志。LPS刺激后兩種DC亞型均顯著上調CD40、CD80和CD86等共刺激分子以及MHC-Ⅰ和MHC-Ⅱ類分子的表達。經不同的TLR激動劑刺激后,cDC和pDC均表達較高水平的IL-6和TfNF-α,且pDC分泌TNF-α的水平顯著高于cDC,但是它們均顯著低于在GM-DC中的表達水平：cDC和pDC經各種TLR激動劑刺激均未表達IL-10、MCP-1,而GM-DC中這兩種因子的表達水平較高；pDC經CpG刺激后分泌IFN-α的水平顯著高于GM-DC和cDC；此外,三種DC亞型均未明顯表達IFN-γ細胞因子。另外,cDC和pDC亞型均具備一定的活化T細胞并促進其分泌IFN-γ和IL-4 T細胞相關細胞因子的能力,但是cDC表現出更強的刺激活化能力。以上結果顯示成功獲得了具備良好生物學功能的cDC和pDC亞型,為下一步分析其在分枝桿菌免疫應答的作用奠定基礎。2、小鼠骨髓源DC亞型在BCG體外感染早期的免疫應答特性分析對Flt3L誘導的FL-cDC和FL-pDC亞型在rBCG-GFP體外感染早期的免疫應答特性進行分析,包括細胞的感染率、活化成熟、細胞因子分泌和特異性抗原提呈能力等。通過激光共聚焦顯微鏡可以在小部分cDC和pDC內觀察到細菌,但是大部分細胞中并沒有細菌；FCM分析也顯示兩種DC亞型的GFP陽性率較低,說明BCG對它們的感染水平較低。但是大部分FL-cDC和FL-pDC在感染后4 h即表達高水平的CD40、CD80、CD86共刺激分子和MHC-Ⅰ、MHC-Ⅱ類分子,且在感染后12 h達到最高峰。對FL-cDC和FL-pDC培養(yǎng)上清中IL-6和TNF-α含量的分析顯示,在感染后4 h,FL-pDC中IL-6和TNF-α的表達量均顯著高于FL-cDC,且持續(xù)至12 h,而FL-cDC中IL-6和]TNF-α的表達量在12 h達到高峰,感染后12 h兩種亞型中IL-6和TNF-α的表達量均開始下降。對BCG相關抗原Ag85A多肽的體外抗原提呈試驗顯示, FL-cDC和FL-pDC亞型均能特異性提呈該多肽,且孵育12 h的提呈能力最強,但是FL-pDC的抗原提呈能力在整個過程中均弱于FL-cDC。這些結果顯示FL-cDC和FL-pDC在體外均可以識別并吞噬BCG,刺激細胞高度活化,表達高水平的共刺激分子和MHC-Ⅰ類和MHC-Ⅱ類分子,同時分泌大量的IL-6和TNF-α細胞因子,并具備提呈BCG相關抗原的能力。3、小鼠脾臟不同DC亞型在機體抗BCG免疫應答中的作用BCG免疫小鼠,對其脾臟pDC和cDC以及CD8+ cDC和CD8- cDC亞型在感染早期的免疫應答特性進行分析,同時對持續(xù)感染過程中脾臟cDC和pDC胞內BCG的生存及細胞的殺菌機制進行分析。首先分析了BCG免疫初期(48 h內)不同脾臟DC亞型的免疫應答特性,FCM分析顯示脾臟pDC的感染率顯著高于cDC,且表現出更高的敏感性；免疫24 h內CD8+ cDC和CD8- cDC的感染率基本相同,隨后CD8+ cDC的感染率逐漸下降,而CD8- cDC的感染率則逐漸提高。與未免疫對照相比,BCG免疫后pDC、CD8+ cDC和CD8-cDC中CD40、CD80、CD86和MHC-Ⅱ類分子陽性的細胞比例均增加,且在免疫后4 h達到最高峰,隨后下降；進一步分析顯示pDC和CD8+ cDC中不僅上述表面分子陽性的細胞比例增加,這些陽性細胞表面各分子的表達強度也增加,而CD8- cDC中相應分子的表達強度并未增加。此外,BCG相關抗原Ag85A蛋白免疫小鼠后,脾臟cDC和pDC均表現出抗原提呈能力,且在免疫后4 h的提呈水平最強,隨后迅速下降,其中cDC比pDC表現出更強的抗原提呈能力。同時,BCG感染早期脾臟cDC相比pDC表達更高水平的IL-12,而pDC中TNF-α的表達量更高。在BCG的持續(xù)感染過程中,脾臟pDC和CD8+ cDC的rBCG-GFP陽性率在感染的不同階段逐漸降低至本底水平,而CD8- cDC中的rBCG-GFP陽性率雖然也有下降,但最終穩(wěn)定在約1%的水平。對免疫后不同階段脾臟cDC和pDC胞內BCG的CFU計數也表現出類似的結果,即pDC胞內細菌數量逐漸下降,但至免疫后60 d胞內仍有細菌存在；而cDC胞內細菌的數量雖然也有下降,但自免疫15 d后即基本保持不變,逐漸顯著高于pDC中的細菌數量；另外,這些長期存在于胞內的細菌能持續(xù)刺激宿主細胞高表達CD40、CD80、CD86和MHC-Ⅱ類分子。在此基礎上,對DC可能的殺菌機制進行探討,在免疫后7 d脾臟cDC比pDC表現出更高的胞內總N0水平,而兩種DC在BCG免疫后均表現出胞內LC3-Ⅱ表達量升高以及GFP-LC3B和RFP-62在胞內的聚集,但是pDC比cDC的表達量更高,且持續(xù)的時間更長,這顯示在cDC和pDC胞內發(fā)揮主要殺菌作用的機制可能各有側重。4、小鼠骨髓源DC亞型在BCG體外感染早期的基因表達譜分析通過cDNA表達譜芯片對小鼠FL-cDC和FL-pDC亞型在BCG感染早期的差異基因表達譜進行分析,以期從分子水平對兩種DC亞型在抗BCG免疫應答的作用進行更廣泛的探討。通過體外誘導并分選FL-cDC和FL-pDC,經BCG感染4 h,通過芯片雜交,分析兩種DC亞型在感染后4 h和24 h相對未感染細胞(分別定義為4c/0c、24c/0c、4p/0p和24p/0p四組數據)的基因表達譜。本研究將P (corr)的值小于或等于0.05且FC大于或等于2的基因定義為上調差異表達,將P (corr)的值小于或等于0.05且FC小于或等于-2的基因定義為下調差異表達。芯片的分析結果顯示,4c/0c組的差異表達基因1974個,其中上調表達1305個,下調表達669個；24c/0c組的差異表達基因1402個,其中上調表達765個,下調表達637個；4p/0p組的差異表達基因1100個,其中上調表達736個,下調表達364個：24p/0p組的差異表達基因1299個,其中上調表達873個,下調表達426個。通過DAVID在線分析軟件,對這些差異基因進行Gene Ontology (GO)的分子功能(Molecular function)分類分析,顯示其主要與各種物質的結合、轉錄因子活性、細胞因子和趨化因子活性、細胞因子和趨化因子受體活性等諸多GO條目有關,且不同組數據之間GO條目的種類和富集程度存在著廣泛差異。信號通路(KEGG pathway)分析顯示這些差異基因主要涉及細胞因子-細胞因子受體相互作用、細胞外基質-受體相互作用、粘著連接、TLR信號通路和NLR信號通路以及某些癌癥相關的信號通路等,其種類和富集程度在不同組數據間同樣也存在差異。重點分析了細胞因子和細胞因子受體相關基因的表達,結果顯示兩種DC亞型在感染早期均有大量的細胞因子和趨化因子活性相關基因上調表達,但相關受體多下調表達,其中腫瘤壞死因子及其受體超家族在感染的過程中發(fā)揮重要的免疫調節(jié)作用。
[Abstract]:Tuberculosis (TB) is a chronic zoonotic infectious disease caused mainly by Mycobacterium tuberculosis (MTB). TB is an ancient disease. It was discovered in Egypt more than 5,000 years ago. Since the discovery of the pathogen of TB in the late 19th century, humans have treated it. However, TB is still the most important bacterial infectious disease in the world. About one-third of the world's population has been infected by TB pathogens, with 8 million new TB cases and 1.3 million deaths each year. MTB is one of the largest single-factor fatal diseases and one of the world's major disease burdens as intracellular bacteria. Dendritic cells (DCs), as the most powerful antigen presenting cells in vivo, trigger the initial T cell immune response and secrete IL-12 and other cytokines by presenting antigens. Anti-TB immune responses play a key role as a bridge between innate and acquired immune responses. However, DCs are highly heterogeneous populations composed of many phenotypic and functional subtypes. In steady-state mice, DCs in the spleen mainly include plasma cell DC (pDCs) and classical DC (cDC), which in turn include C. Subtypes D8+cDC and CD8+cDC. Further study of specific immune functions of different DC subtypes in anti-TB immune response will greatly promote understanding of the process of anti-TB immune response. However, due to the scarcity of DCs and difficulties in subtype sorting and preparation, the role of different DC subtypes in the immune response to Mycobacterium In this study, the immune response characteristics of different subtypes of dendritic cells to BCG infection in vivo and in vitro were analyzed, and the gene expression profiles of different DC subtypes in the early stage of BCG infection in vitro were compared in order to increase the role of different DC subtypes in the immune response to Mycobacterium. To understand. 1. Induction, identification and characterization of mouse bone marrow derived DC subtypes in vitro. The cDC and pDC subtypes of DCs were prepared from mouse bone marrow hematopoietic stem cells induced by mouse Flt3L protein in vitro. The morphological changes of cells during induction were observed and the expression of characteristic molecular markers was analyzed. The expression profiles of cytokines and the ability to induce T cell proliferation were analyzed. Flow cytometry (FCM) analysis showed that the positive rate of CD11c was over 60% after 9 days of culture in vitro. Typical dendritic morphology was observed under optical microscope after LPS activation. After induction, CD11c+CD45RA-and CD11c+CD45RA+ phenotypes were obtained. Characteristic cDC and pDC subtypes, and cDC can be further divided into CD24 high CD11 blow and CD24 low CDbhigh subtypes. At the same time, pDC also specifically expresses mPDCA-1 molecular markers. After LPS stimulation, the expression of CD40, CD80 and CD86 co-stimulatory molecules, MHC-I and MHC-II molecules were significantly up-regulated. After stimulation by different TLR agonists, cDC-I and MHC-II molecules were up-regulated. Both pDC and pDC expressed higher levels of IL-6 and TfNF-alpha, and the levels of TNF-alpha secreted by pDC were significantly higher than those of cDC, but they were significantly lower than those in GM-DC. Both cDC and pDC did not express IL-10 and MCP-1 after stimulation with various TLR agonists, while the levels of IFN-alpha secreted by pDC were significantly higher after stimulation with CpG. In addition, both cDC and pDC subtypes have the ability to activate T cells and promote their secretion of IFN-gamma and IL-4 T cell-related cytokines, but cDC has a stronger stimulating and activating ability. These results show that cDC has been successfully obtained with good biological characteristics. Functional cDC and pDC subtypes lay the foundation for further analysis of their role in Mycobacterium immune response. 2. Immune response characteristics of mouse bone marrow-derived DC subtypes in the early stage of BCG infection in vitro were analyzed, including the infection rate of cells. Bacteria can be observed in a small number of cDC and pDC by laser confocal microscopy, but there are no bacteria in most cells. FCM analysis also shows that the positive rate of GFP in the two DC subtypes is low, indicating that BCG has a low level of infection to them. The expression levels of CD40, CD80, CD86 costimulatory molecules and MHC-I, MHC-II molecules in the supernatants of FL-cDC and FL-pDC reached the highest level at 4 h after infection, and reached the peak at 12 h after infection. The expression levels of IL-6 and]TNF-alpha in-cDC reached the peak at 12 h and began to decrease at 12 h after infection. Antigen presenting test of BCG-associated antigen Ag85A polypeptide in vitro showed that both FL-cDC and FL-pDC subtypes could specifically presenting the polypeptide, and the presenting ability of FL-pDC was the strongest at 12 h after incubation. These results indicate that FL-cDC and FL-pDC can recognize and phagocytize BCG in vitro, stimulate cell activation, express high levels of costimulatory molecules and MHC-I and MHC-II molecules, secrete a large number of IL-6 and TNF-a cytokines, and have the ability to present BCG-related antigens. 3, mice The role of different splenic DC subtypes in the body's anti-BCG immune response was investigated in mice immunized with BCG. The immune response characteristics of splenic pDC and cDC, CD8+cDC and CD8-cDC subtypes in the early stage of infection were analyzed. The survival and bactericidal mechanism of splenic cDC and pDC intracellular BCG during persistent infection were analyzed. FCM analysis showed that the infection rate of splenic pDC was significantly higher than that of cDC, and the infection rate of CD8+cDC and CD8-cDC were basically the same within 24 hours of immunization, and then the infection rate of CD8+cDC gradually decreased, while the infection rate of CD8-cDC gradually increased. Compared with BCG, the percentage of CD40, CD80, CD86 and MHC-II positive cells in pDC, CD8+cDC and CD8-cDC increased after BCG immunization, and reached the peak at 4 h after BCG immunization, then decreased; further analysis showed that the proportion of positive cells in pDC and CD8+cDC not only increased, but also the expression intensity of the molecules on the surface of these positive cells. In addition, after immunization with Ag85A protein, both spleen cDC and pDC exhibited antigen presenting ability, and the presenting level was the strongest at 4 h after immunization, and then decreased rapidly. Among them, cDC showed stronger antigen presenting ability than pDC. The positive rate of rBCG-GFP in spleen pDC and CD8+cDC gradually decreased to the background level in different stages of infection, while the positive rate of rBCG-GFP in CD8-cDC also decreased, but eventually stabilized at about 1% level. Similar results were found in the CFU counts of spleen cDC and intracellular BCG of pDC at different stages, that is, the number of bacteria in pDC decreased gradually, but there were still bacteria in pDC at 60 days after immunization, while the number of bacteria in cDC decreased, but remained unchanged after 15 days of immunization, and the number of bacteria in pDC was gradually higher than that in pDC. On this basis, the possible bactericidal mechanisms of DCs were discussed. Spleen cDC showed higher intracellular total N0 levels than pDC at 7 days after immunization, while both DCs showed increased intracellular LC3-II expression and GFP-LC3B after BCG immunization. The expression of pDC and RFP-62 was higher and lasted longer than that of cDC, suggesting that the main bactericidal mechanisms of cDC and pDC might be different. 4. Gene expression profiles of mouse bone marrow-derived DC subtypes in the early stage of BCG infection in vitro were analyzed by cDNA expression profiling chip for FL-cDC and FL-pDC subtypes in BC. Differential gene expression profiles in the early stage of G infection were analyzed in order to explore the role of the two DC subtypes in the anti-BCG immune response at the molecular level. The gene expression profiles of 4c/0c, 24c/0c, 4p/0p, and 24p/0p groups were defined as up-regulation of differential expression. The gene with P (corr) value less than or equal to 0.05 and FC greater than or equal to 2 was defined as up-regulation of differential expression. The gene with P (corr) value less than or equal to 0.05 and FC less than or equal to - 2 was defined as down-regulation of differential expression. Among the 1974 differentially expressed genes, 1 305 were up-regulated and 669 down-regulated; 1 402 were down-regulated in 24c/0c group, of which 765 were up-regulated and 637 were down-regulated; 1 100 were down-regulated in 4p/0p group, of which 736 were up-regulated and 364 were down-regulated: 1 299 were down-regulated in 24p/0p group, of which 873 were up-regulated. Gene Ontology (GO) molecular function was classified and analyzed by DAVID online analysis software. The results showed that these genes were mainly combined with various substances, transcription factor activity, cytokine and chemokine activity, cytokine and chemokine receptor activity and so on. KEGG pathway analysis showed that these genes were mainly involved in cytokine-cytokine receptor interaction, extracellular matrix-receptor interaction, adhesion junction, TLR signaling pathway and NLR signaling pathway, and some cancers. The expression of cytokine and cytokine receptor-related genes was analyzed emphatically. The results showed that there were a large number of cytokine and chemokine-related genes up-regulated and down-regulated in both DC subtypes in the early stage of infection. Tumor necrosis factor and tumor necrosis factor receptor superfamily play an important role in the immunoregulation of infection.
【學位授予單位】：揚州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392

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相關博士學位論文 前1條

1 劉曼莉;豬鏈球菌2型感染宿主后宿主表達譜的分析研究[D];華中農業(yè)大學;2011年

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