【摘要】：結(jié)核病(Tuberculosis, TB)是主要由結(jié)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(xiàn)了該病的存在,自19世紀(jì)末發(fā)現(xiàn)TB的致病原以來(lái),人類對(duì)它們的研究已經(jīng)持續(xù)了一百多年。然而,TB仍是目前世界上最重要的細(xì)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(wàn)TB新增病例和130萬(wàn)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(fù)擔(dān)之一MTB作為胞內(nèi)菌,其引起的慢性傳染多數(shù)處于潛伏感染狀態(tài),機(jī)體產(chǎn)生的抗菌免疫應(yīng)答決定疾病的發(fā)生和發(fā)展。樹(shù)突狀細(xì)胞(Dendritic cells, DCs)作為體內(nèi)功能最強(qiáng)的抗原提呈細(xì)胞,通過(guò)提呈抗原觸發(fā)初始T細(xì)胞免疫應(yīng)答并分泌IL-12等細(xì)胞因子的方式在機(jī)體抗TB免疫應(yīng)答中發(fā)揮關(guān)鍵作用,是聯(lián)系天然免疫和獲得性免疫應(yīng)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(zhì)性群體,穩(wěn)態(tài)時(shí)小鼠脾臟DCs主要包括漿細(xì)胞DC(plasmacytoid DCs, pDCs)和經(jīng)典DC (conventional DC, cDC),后者又包括CD8+ cDC和CD8+ cDC等亞型。對(duì)不同DC亞型在抗TB免疫應(yīng)答中的具體免疫功能的深入研究將極大促進(jìn)對(duì)機(jī)體抗TB應(yīng)答過(guò)程的了解。然而,由于DCs數(shù)量稀少以及亞型分選和制備困難等原因,目前對(duì)不同DC亞型在機(jī)體抗分枝桿菌免疫應(yīng)答中的作用還所知甚少。本研究在體內(nèi)和體外條件下對(duì)小鼠不同亞型樹(shù)突狀細(xì)胞在BCG感染過(guò)程中的免疫應(yīng)答特性進(jìn)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(dá)譜差異,以期增加對(duì)不同DC亞型在機(jī)體抗分枝桿菌免疫應(yīng)答中的作用的了解。1、小鼠骨髓源DC亞型的體外誘導(dǎo)、鑒定及其生物學(xué)特性分析通過(guò)小鼠Flt3L蛋白體外誘導(dǎo)骨髓造血干細(xì)胞制備DCs的cDC和pDC亞型,觀察誘導(dǎo)過(guò)程中細(xì)胞形態(tài)學(xué)變化,分析其特征性分子標(biāo)志的表達(dá),并對(duì)其活化前后共刺激分子的表達(dá)、細(xì)胞因子表達(dá)譜以及誘導(dǎo)T細(xì)胞增殖的能力等功能特征進(jìn)行分析。流式細(xì)胞術(shù)(FCM)分析顯示體外培養(yǎng)9 d后CD11c的陽(yáng)性率可達(dá)60%以上,經(jīng)LPS活化后在光學(xué)顯微鏡下可觀察到典型的樹(shù)突狀形態(tài)。誘導(dǎo)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(jìn)一步分為CD24highCD11blow和CD24lowCDbhigh兩個(gè)亞型,同時(shí),pDC也特異性表達(dá)mPDCA-1分子標(biāo)志。LPS刺激后兩種DC亞型均顯著上調(diào)CD40、CD80和CD86等共刺激分子以及MHC-Ⅰ和MHC-Ⅱ類分子的表達(dá)。經(jīng)不同的TLR激動(dòng)劑刺激后,cDC和pDC均表達(dá)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(dá)水平：cDC和pDC經(jīng)各種TLR激動(dòng)劑刺激均未表達(dá)IL-10、MCP-1,而GM-DC中這兩種因子的表達(dá)水平較高；pDC經(jīng)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(dá)IFN-γ細(xì)胞因子。另外,cDC和pDC亞型均具備一定的活化T細(xì)胞并促進(jìn)其分泌IFN-γ和IL-4 T細(xì)胞相關(guān)細(xì)胞因子的能力,但是cDC表現(xiàn)出更強(qiáng)的刺激活化能力。以上結(jié)果顯示成功獲得了具備良好生物學(xué)功能的cDC和pDC亞型,為下一步分析其在分枝桿菌免疫應(yīng)答的作用奠定基礎(chǔ)。2、小鼠骨髓源DC亞型在BCG體外感染早期的免疫應(yīng)答特性分析對(duì)Flt3L誘導(dǎo)的FL-cDC和FL-pDC亞型在rBCG-GFP體外感染早期的免疫應(yīng)答特性進(jìn)行分析,包括細(xì)胞的感染率、活化成熟、細(xì)胞因子分泌和特異性抗原提呈能力等。通過(guò)激光共聚焦顯微鏡可以在小部分cDC和pDC內(nèi)觀察到細(xì)菌,但是大部分細(xì)胞中并沒(méi)有細(xì)菌；FCM分析也顯示兩種DC亞型的GFP陽(yáng)性率較低,說(shuō)明BCG對(duì)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(dá)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(dá)到最高峰。對(duì)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(dá)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(dá)量在12 h達(dá)到高峰,感染后12 h兩種亞型中IL-6和TNF-α的表達(dá)量均開(kāi)始下降。對(duì)BCG相關(guān)抗原Ag85A多肽的體外抗原提呈試驗(yàn)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(qiáng),但是FL-pDC的抗原提呈能力在整個(gè)過(guò)程中均弱于FL-cDC。這些結(jié)果顯示FL-cDC和FL-pDC在體外均可以識(shí)別并吞噬BCG,刺激細(xì)胞高度活化,表達(dá)高水平的共刺激分子和MHC-Ⅰ類和MHC-Ⅱ類分子,同時(shí)分泌大量的IL-6和TNF-α細(xì)胞因子,并具備提呈BCG相關(guān)抗原的能力。3、小鼠脾臟不同DC亞型在機(jī)體抗BCG免疫應(yīng)答中的作用BCG免疫小鼠,對(duì)其脾臟pDC和cDC以及CD8+ cDC和CD8- cDC亞型在感染早期的免疫應(yīng)答特性進(jìn)行分析,同時(shí)對(duì)持續(xù)感染過(guò)程中脾臟cDC和pDC胞內(nèi)BCG的生存及細(xì)胞的殺菌機(jī)制進(jìn)行分析。首先分析了BCG免疫初期(48 h內(nèi))不同脾臟DC亞型的免疫應(yīng)答特性,FCM分析顯示脾臟pDC的感染率顯著高于cDC,且表現(xiàn)出更高的敏感性；免疫24 h內(nèi)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(duì)照相比,BCG免疫后pDC、CD8+ cDC和CD8-cDC中CD40、CD80、CD86和MHC-Ⅱ類分子陽(yáng)性的細(xì)胞比例均增加,且在免疫后4 h達(dá)到最高峰,隨后下降；進(jìn)一步分析顯示pDC和CD8+ cDC中不僅上述表面分子陽(yáng)性的細(xì)胞比例增加,這些陽(yáng)性細(xì)胞表面各分子的表達(dá)強(qiáng)度也增加,而CD8- cDC中相應(yīng)分子的表達(dá)強(qiáng)度并未增加。此外,BCG相關(guān)抗原Ag85A蛋白免疫小鼠后,脾臟cDC和pDC均表現(xiàn)出抗原提呈能力,且在免疫后4 h的提呈水平最強(qiáng),隨后迅速下降,其中cDC比pDC表現(xiàn)出更強(qiáng)的抗原提呈能力。同時(shí),BCG感染早期脾臟cDC相比pDC表達(dá)更高水平的IL-12,而pDC中TNF-α的表達(dá)量更高。在BCG的持續(xù)感染過(guò)程中,脾臟pDC和CD8+ cDC的rBCG-GFP陽(yáng)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(yáng)性率雖然也有下降,但最終穩(wěn)定在約1%的水平。對(duì)免疫后不同階段脾臟cDC和pDC胞內(nèi)BCG的CFU計(jì)數(shù)也表現(xiàn)出類似的結(jié)果,即pDC胞內(nèi)細(xì)菌數(shù)量逐漸下降,但至免疫后60 d胞內(nèi)仍有細(xì)菌存在；而cDC胞內(nèi)細(xì)菌的數(shù)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(xì)菌數(shù)量；另外,這些長(zhǎng)期存在于胞內(nèi)的細(xì)菌能持續(xù)刺激宿主細(xì)胞高表達(dá)CD40、CD80、CD86和MHC-Ⅱ類分子。在此基礎(chǔ)上,對(duì)DC可能的殺菌機(jī)制進(jìn)行探討,在免疫后7 d脾臟cDC比pDC表現(xiàn)出更高的胞內(nèi)總N0水平,而兩種DC在BCG免疫后均表現(xiàn)出胞內(nèi)LC3-Ⅱ表達(dá)量升高以及GFP-LC3B和RFP-62在胞內(nèi)的聚集,但是pDC比cDC的表達(dá)量更高,且持續(xù)的時(shí)間更長(zhǎng),這顯示在cDC和pDC胞內(nèi)發(fā)揮主要?dú)⒕饔玫臋C(jī)制可能各有側(cè)重。4、小鼠骨髓源DC亞型在BCG體外感染早期的基因表達(dá)譜分析通過(guò)cDNA表達(dá)譜芯片對(duì)小鼠FL-cDC和FL-pDC亞型在BCG感染早期的差異基因表達(dá)譜進(jìn)行分析,以期從分子水平對(duì)兩種DC亞型在抗BCG免疫應(yīng)答的作用進(jìn)行更廣泛的探討。通過(guò)體外誘導(dǎo)并分選FL-cDC和FL-pDC,經(jīng)BCG感染4 h,通過(guò)芯片雜交,分析兩種DC亞型在感染后4 h和24 h相對(duì)未感染細(xì)胞(分別定義為4c/0c、24c/0c、4p/0p和24p/0p四組數(shù)據(jù))的基因表達(dá)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(diào)差異表達(dá),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(diào)差異表達(dá)。芯片的分析結(jié)果顯示,4c/0c組的差異表達(dá)基因1974個(gè),其中上調(diào)表達(dá)1305個(gè),下調(diào)表達(dá)669個(gè)；24c/0c組的差異表達(dá)基因1402個(gè),其中上調(diào)表達(dá)765個(gè),下調(diào)表達(dá)637個(gè)；4p/0p組的差異表達(dá)基因1100個(gè),其中上調(diào)表達(dá)736個(gè),下調(diào)表達(dá)364個(gè)：24p/0p組的差異表達(dá)基因1299個(gè),其中上調(diào)表達(dá)873個(gè),下調(diào)表達(dá)426個(gè)。通過(guò)DAVID在線分析軟件,對(duì)這些差異基因進(jìn)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(zhì)的結(jié)合、轉(zhuǎn)錄因子活性、細(xì)胞因子和趨化因子活性、細(xì)胞因子和趨化因子受體活性等諸多GO條目有關(guān),且不同組數(shù)據(jù)之間GO條目的種類和富集程度存在著廣泛差異。信號(hào)通路(KEGG pathway)分析顯示這些差異基因主要涉及細(xì)胞因子-細(xì)胞因子受體相互作用、細(xì)胞外基質(zhì)-受體相互作用、粘著連接、TLR信號(hào)通路和NLR信號(hào)通路以及某些癌癥相關(guān)的信號(hào)通路等,其種類和富集程度在不同組數(shù)據(jù)間同樣也存在差異。重點(diǎn)分析了細(xì)胞因子和細(xì)胞因子受體相關(guān)基因的表達(dá),結(jié)果顯示兩種DC亞型在感染早期均有大量的細(xì)胞因子和趨化因子活性相關(guān)基因上調(diào)表達(dá),但相關(guān)受體多下調(diào)表達(dá),其中腫瘤壞死因子及其受體超家族在感染的過(guò)程中發(fā)揮重要的免疫調(diào)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(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)博士學(xué)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(dá)譜的分析研究[D];華中農(nóng)業(yè)大學(xué);2011年

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本文編號(hào)：2187081