小鼠树突状细胞亚型cDC和pDC在BCG免疫应答中的作用
发布时间：2018-08-17 08:43
【摘要】：结核病(Tuberculosis, TB)是主要由结核分枝杆菌(Mycobacterium tuberculosis, MTB)引起的一类以呼吸系统感染为主的慢性人兽共患传染病。TB是一种古老的疾病,在5000余年前的埃及就发现了该病的存在,自19世纪末发现TB的致病原以来,人类对它们的研究已经持续了一百多年。然而,TB仍是目前世界上最重要的细菌性传染病,全球约I/3人口已被TB病原菌感染,每年有约800万TB新增病例和130万死亡病例,是人类传染病中最大的单因素致死疾病和全球主要疾病负担之一MTB作为胞内菌,其引起的慢性传染多数处于潜伏感染状态,机体产生的抗菌免疫应答决定疾病的发生和发展。树突状细胞(Dendritic cells, DCs)作为体内功能最强的抗原提呈细胞,通过提呈抗原触发初始T细胞免疫应答并分泌IL-12等细胞因子的方式在机体抗TB免疫应答中发挥关键作用,是联系天然免疫和获得性免疫应答的桥梁。然而,DCs是由许多表型和功能不同的亚型组成的高度异质性群体,稳态时小鼠脾脏DCs主要包括浆细胞DC(plasmacytoid DCs, pDCs)和经典DC (conventional DC, cDC),后者又包括CD8+ cDC和CD8+ cDC等亚型。对不同DC亚型在抗TB免疫应答中的具体免疫功能的深入研究将极大促进对机体抗TB应答过程的了解。然而,由于DCs数量稀少以及亚型分选和制备困难等原因,目前对不同DC亚型在机体抗分枝杆菌免疫应答中的作用还所知甚少。本研究在体内和体外条件下对小鼠不同亚型树突状细胞在BCG感染过程中的免疫应答特性进行了分析,并比较了不同DC亚型在BCG体外感染早期的基因表达谱差异,以期增加对不同DC亚型在机体抗分枝杆菌免疫应答中的作用的了解。1、小鼠骨髓源DC亚型的体外诱导、鉴定及其生物学特性分析通过小鼠Flt3L蛋白体外诱导骨髓造血干细胞制备DCs的cDC和pDC亚型,观察诱导过程中细胞形态学变化,分析其特征性分子标志的表达,并对其活化前后共刺激分子的表达、细胞因子表达谱以及诱导T细胞增殖的能力等功能特征进行分析。流式细胞术(FCM)分析显示体外培养9 d后CD11c的阳性率可达60%以上,经LPS活化后在光学显微镜下可观察到典型的树突状形态。诱导后获得了具备CD11c+CD45RA-和CD11c+CD45RA+表型特征的cDC和pDC亚型,且cDC可进一步分为CD24highCD11blow和CD24lowCDbhigh两个亚型,同时,pDC也特异性表达mPDCA-1分子标志。LPS刺激后两种DC亚型均显著上调CD40、CD80和CD86等共刺激分子以及MHC-Ⅰ和MHC-Ⅱ类分子的表达。经不同的TLR激动剂刺激后,cDC和pDC均表达较高水平的IL-6和TfNF-α,且pDC分泌TNF-α的水平显著高于cDC,但是它们均显著低于在GM-DC中的表达水平：cDC和pDC经各种TLR激动剂刺激均未表达IL-10、MCP-1,而GM-DC中这两种因子的表达水平较高；pDC经CpG刺激后分泌IFN-α的水平显著高于GM-DC和cDC；此外,三种DC亚型均未明显表达IFN-γ细胞因子。另外,cDC和pDC亚型均具备一定的活化T细胞并促进其分泌IFN-γ和IL-4 T细胞相关细胞因子的能力,但是cDC表现出更强的刺激活化能力。以上结果显示成功获得了具备良好生物学功能的cDC和pDC亚型,为下一步分析其在分枝杆菌免疫应答的作用奠定基础。2、小鼠骨髓源DC亚型在BCG体外感染早期的免疫应答特性分析对Flt3L诱导的FL-cDC和FL-pDC亚型在rBCG-GFP体外感染早期的免疫应答特性进行分析,包括细胞的感染率、活化成熟、细胞因子分泌和特异性抗原提呈能力等。通过激光共聚焦显微镜可以在小部分cDC和pDC内观察到细菌,但是大部分细胞中并没有细菌；FCM分析也显示两种DC亚型的GFP阳性率较低,说明BCG对它们的感染水平较低。但是大部分FL-cDC和FL-pDC在感染后4 h即表达高水平的CD40、CD80、CD86共刺激分子和MHC-Ⅰ、MHC-Ⅱ类分子,且在感染后12 h达到最高峰。对FL-cDC和FL-pDC培养上清中IL-6和TNF-α含量的分析显示,在感染后4 h,FL-pDC中IL-6和TNF-α的表达量均显著高于FL-cDC,且持续至12 h,而FL-cDC中IL-6和]TNF-α的表达量在12 h达到高峰,感染后12 h两种亚型中IL-6和TNF-α的表达量均开始下降。对BCG相关抗原Ag85A多肽的体外抗原提呈试验显示, FL-cDC和FL-pDC亚型均能特异性提呈该多肽,且孵育12 h的提呈能力最强,但是FL-pDC的抗原提呈能力在整个过程中均弱于FL-cDC。这些结果显示FL-cDC和FL-pDC在体外均可以识别并吞噬BCG,刺激细胞高度活化,表达高水平的共刺激分子和MHC-Ⅰ类和MHC-Ⅱ类分子,同时分泌大量的IL-6和TNF-α细胞因子,并具备提呈BCG相关抗原的能力。3、小鼠脾脏不同DC亚型在机体抗BCG免疫应答中的作用BCG免疫小鼠,对其脾脏pDC和cDC以及CD8+ cDC和CD8- cDC亚型在感染早期的免疫应答特性进行分析,同时对持续感染过程中脾脏cDC和pDC胞内BCG的生存及细胞的杀菌机制进行分析。首先分析了BCG免疫初期(48 h内)不同脾脏DC亚型的免疫应答特性,FCM分析显示脾脏pDC的感染率显著高于cDC,且表现出更高的敏感性；免疫24 h内CD8+ cDC和CD8- cDC的感染率基本相同,随后CD8+ cDC的感染率逐渐下降,而CD8- cDC的感染率则逐渐提高。与未免疫对照相比,BCG免疫后pDC、CD8+ cDC和CD8-cDC中CD40、CD80、CD86和MHC-Ⅱ类分子阳性的细胞比例均增加,且在免疫后4 h达到最高峰,随后下降；进一步分析显示pDC和CD8+ cDC中不仅上述表面分子阳性的细胞比例增加,这些阳性细胞表面各分子的表达强度也增加,而CD8- cDC中相应分子的表达强度并未增加。此外,BCG相关抗原Ag85A蛋白免疫小鼠后,脾脏cDC和pDC均表现出抗原提呈能力,且在免疫后4 h的提呈水平最强,随后迅速下降,其中cDC比pDC表现出更强的抗原提呈能力。同时,BCG感染早期脾脏cDC相比pDC表达更高水平的IL-12,而pDC中TNF-α的表达量更高。在BCG的持续感染过程中,脾脏pDC和CD8+ cDC的rBCG-GFP阳性率在感染的不同阶段逐渐降低至本底水平,而CD8- cDC中的rBCG-GFP阳性率虽然也有下降,但最终稳定在约1%的水平。对免疫后不同阶段脾脏cDC和pDC胞内BCG的CFU计数也表现出类似的结果,即pDC胞内细菌数量逐渐下降,但至免疫后60 d胞内仍有细菌存在；而cDC胞内细菌的数量虽然也有下降,但自免疫15 d后即基本保持不变,逐渐显著高于pDC中的细菌数量；另外,这些长期存在于胞内的细菌能持续刺激宿主细胞高表达CD40、CD80、CD86和MHC-Ⅱ类分子。在此基础上,对DC可能的杀菌机制进行探讨,在免疫后7 d脾脏cDC比pDC表现出更高的胞内总N0水平,而两种DC在BCG免疫后均表现出胞内LC3-Ⅱ表达量升高以及GFP-LC3B和RFP-62在胞内的聚集,但是pDC比cDC的表达量更高,且持续的时间更长,这显示在cDC和pDC胞内发挥主要杀菌作用的机制可能各有侧重。4、小鼠骨髓源DC亚型在BCG体外感染早期的基因表达谱分析通过cDNA表达谱芯片对小鼠FL-cDC和FL-pDC亚型在BCG感染早期的差异基因表达谱进行分析,以期从分子水平对两种DC亚型在抗BCG免疫应答的作用进行更广泛的探讨。通过体外诱导并分选FL-cDC和FL-pDC,经BCG感染4 h,通过芯片杂交,分析两种DC亚型在感染后4 h和24 h相对未感染细胞(分别定义为4c/0c、24c/0c、4p/0p和24p/0p四组数据)的基因表达谱。本研究将P (corr)的值小于或等于0.05且FC大于或等于2的基因定义为上调差异表达,将P (corr)的值小于或等于0.05且FC小于或等于-2的基因定义为下调差异表达。芯片的分析结果显示,4c/0c组的差异表达基因1974个,其中上调表达1305个,下调表达669个；24c/0c组的差异表达基因1402个,其中上调表达765个,下调表达637个；4p/0p组的差异表达基因1100个,其中上调表达736个,下调表达364个：24p/0p组的差异表达基因1299个,其中上调表达873个,下调表达426个。通过DAVID在线分析软件,对这些差异基因进行Gene Ontology (GO)的分子功能(Molecular function)分类分析,显示其主要与各种物质的结合、转录因子活性、细胞因子和趋化因子活性、细胞因子和趋化因子受体活性等诸多GO条目有关,且不同组数据之间GO条目的种类和富集程度存在着广泛差异。信号通路(KEGG pathway)分析显示这些差异基因主要涉及细胞因子-细胞因子受体相互作用、细胞外基质-受体相互作用、粘着连接、TLR信号通路和NLR信号通路以及某些癌症相关的信号通路等,其种类和富集程度在不同组数据间同样也存在差异。重点分析了细胞因子和细胞因子受体相关基因的表达,结果显示两种DC亚型在感染早期均有大量的细胞因子和趋化因子活性相关基因上调表达,但相关受体多下调表达,其中肿瘤坏死因子及其受体超家族在感染的过程中发挥重要的免疫调节作用。
[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.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
本文编号：2187081
[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.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
【参考文献】
相关博士学位论文 前1条
1 刘曼莉;猪链球菌2型感染宿主后宿主表达谱的分析研究[D];华中农业大学;2011年
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