鸡NLRC5基因在免疫反应中调控机理的初步研究

发布时间：2018-03-13 20:25

本文选题：NLRC5　切入点：白痢沙门氏菌　出处：《扬州大学》2015年博士论文　论文类型：学位论文

【摘要】：NOD样受体家族CARD结构域包含分子5 (NLR family, CARD domain containing 5, NLRC5)在调控天然免疫和细胞免疫中发挥重要作用,到目前为止,NLRC5已经被证实在人和小鼠中可以调控MHCI类分子的表达,并对NF-κB以及I型干扰素分子通路具有抑制性调控作用。白痢沙门氏菌病是我国家禽养殖重要的细菌性疾病之一,该病的爆发给家禽生产造成严重的经济损失。白痢沙门氏菌的隐性感染和携带状态的形成会导致重复发病进而防治困难。白痢沙门氏菌侵入鸡体内后,会抑制MHC Class I以及诱导IL10表达,这一机制是阻断清除的过程从而导致携带状态和持续感染状态的原因之一。鸡NLRC5分子可能与沙门氏菌清除的分子机理有关。截止目前雏鸡感染白痢沙门氏菌后组织水平的高通量研究尚无报道,其他菌株由于致病性和病理机制的差异仅能提供部分参考,因此有必要专门对鸡白痢沙门氏菌感染过程中的表达谱变化进行系统研究,并在此基础上进一步研究家禽NLRC5及相关基因的表达规律以及启动子缺失对NLRC5表达的影响,从而分析其受调控的可能机制,以帮助建立家禽的NLRC5调控的机制模型,更好的理解家禽白痢沙门氏菌隐性感染形成的机制以及细菌性病原清除的机理。研究内容和结果如下。1.第二章第一节,比较不同沙门氏菌菌种感染导致雏鸡的脾脏和盲肠发生的表达谱变化。通过安捷伦鸡全基因组表达谱芯片,分别对7dpi感染了白痢、肠炎还有poly(I:C)的雏鸡脾脏和盲肠进行了表达谱分析,同时检测IL-la, IFNy, IgG和IgM浓度,肠道载菌量和免疫荧光鉴定脾脏载菌量。结果表明,病原刺激激发了系统性沙门氏菌感染的主要特征性免疫反应。在白痢沙门氏菌感染组中,脾脏表达谱富集分析结果显示,差异表达基因主要富集于淋巴细胞的分化和增殖过程,因而呈现出获得性免疫的有效激活状态；其中与Thl淋巴以及NK细胞激活有关的重要分子诸如IL15, CD28等均呈现下调,而与B淋巴细胞活化有关的基因例如IL7呈现上调。这表明细胞免疫的清除作用一定程度上已经受到了抑制,而B细胞所参与的抗体反应被激活。而感染白痢沙门氏菌组的盲肠组织中,下调基因主要与天然性免疫系统有关,表明相关天然性免疫反应或细胞免疫反应活性的减弱,推测可能与抗体的保护作用增强有关。第二章第二节,在证实白痢沙门氏菌感染过程在7dpi造成细胞免疫功能衰弱的基础上,然后利用时间序列表达谱分析白痢沙门氏菌感染雏鸡后2小时、4小时、8小时、24小时、3天、5天、7天、12天、21天脾脏组织中重要的分子事件。同时测定体重、脾脏指数、法氏囊指数、胸腺指数、以及IL-1α、IFNγ、IgG和IgM浓度。通过WGCNA的表型关联分析,以及探针间的共表达特征,韦恩分析等方法挖掘导致隐性感染和携带的差异表达基因。时序表达谱的分析初步探明了在白痢沙门氏菌感染的关键时期脾脏组织中表达谱发生的变化,观察到了天然免疫到体液免疫发展变化过程中脾脏组织中重要的分子事件,包括淋巴细胞的激活,增殖和迁移事件；并鉴定出大量关键调控基因,例如MHC class I B-F, ANXA13, IL18, BF2, TNFRSF13B, BLB2以及CD28等；富集通路中的基因呈现两类高度负相关的功能团；差异表达基因中的节点基因的表达模式与脾脏指数高度正相关；在不同阶段中免疫反应有关的通路表现出与其他信号通路和代谢通路不同的关联特征；差异表达基因大多数均可以定位在有报道的与家禽抗体滴度有关的QTL区域内；NLRC5是差异表达基因；3dpi-5dpi是天然性免疫抑制和细胞免疫激活的关键阶段,也是病原清除的主要时期,该阶段的差异表达基因表达趋势反应了雏鸡对病原做出免疫反应的特征；NLRC5的表达模式表明NLRC5的翻译和激发的下游通路促进了细胞免疫反应和病原的清除作用,但同时可能也受到了负调控因素的调控。第二章第三节,进而基于上述试验提供的表达谱芯片数据,以及NLRC5对MHCI已知的调控作用的假设,开展针对NLRC5在表达谱数据中的共表达分析,尝试找到重要的共表达分子。共表达分析结果显示,NLRC5与TAP1和IL21R高度共表达,其中作为MHCI区域中的分子TAP1可能在受到NF-κB调控的同时也受到NLRC5的影响。2.第三章第一节,鉴于NLRC5基因功能的重要性及其在鸡白痢沙门氏菌感染过程中的表达与调控功能,本研究进一步对鸡NLRC5基因功能进行研究。首先,克隆了鸡NLRC5的全长CDS以及UTR序列,并对其进行细致的生物信息学分析。在组织水平对有报道可能与NLRC5功能相关的基因进行荧光定量的检测和分析。第三章第二节,随后基于克降的片段构建过表达和干扰载体,转染DF1细胞系,并构建稳定表达株。在此基础上首先进行细胞免疫荧光鉴定,然后对细胞进行了LPS刺激,以观察相关基因表达变化的趋势。最后在稳定表达细胞株上对NLRC5以及信号通路中的关键基因的表达模式进行了比较和分析,并提出了NLRC5及重要相关基因的假设调控机制。NLRC5克隆和分析结果显示,鸡NLRC5与哺乳动物NLRC5基因具有一定的同源性,推测可能和哺乳动物该基因功能一致,可以进入核内发挥作用,并受到表观遗传修饰的影响。NLRC5及其信号通路中的关键基因在组织和细胞中的表达研究发现,NF-κB2与NLRC5有显著的线性共表达关系,鸡的STAT1与NLRC5没有明显的共表达特征,推测可能对NLRC5的调控作用较复杂或可能没有调控作用,而STAT3可能作为NF-κB2的负调控因子负反馈调控了NLRC5的表达。综上,除MHCI外,NLRC5可能调控了STAT3和IL18的转录表达。家禽NLRC5可能具有与哺乳动物不同的表达调控机制。3.第四章,为进一步弄清NLRC5受到调控的具体过程,以及进一步证明鸡NLRC5对MHCI的调控作用,进而帮助更好的开展SPI-2 Ⅲ型分泌系统对隐性感染和携菌状态形成作用的研究奠定基础,本研究初步探索了NLRC5起始密码子前4372bp区域不同片段的活性,并分析其潜在的可能调控原理。NLRC5的系列缺失结果证明,NLRC5可能具有2个有活性的启动子区域,并且通过617到1448之间的区域隔离两个启动子的活性；第二个启动子活性要强于第一个启动子活性,并且在该区域内发现了NF-κB等有报道与NLRC5表达调控有密切相关的转录因子的结合基序,1448到4372的片段可能是脾脏组织中的发挥作用的主要启动子,转录表达主要从2108开始的NLRC5分子片段；下游调控可能较为复杂并且下游元件对NLRC5的表达有显著的影响；1-2108的活性仅为1448-4372的50%左右,这表明1448-2108的活性要比1828到2108的活性低,因而推测,1828到2213的活性会进一步增强,甚至超过1448到4372的活性,因此认为NLRC5的核心启动子主要为1828到2213的片段,该区间包含的元件可能有STAT1、NF-κB、P300、MRF2、FAC1、 PAX5、CRFB、CPBP、NF-AT1、MRF-2、AML1、FAC1、CRX、RelA-p65。其中STAT1和NF-κB均于转录起始位点前182bp起始的位置被鉴定出来。未来,研究需确认家禽NLRC5对MHCI调控的机制,通过更进一步的ChIP,乃至ChIP-Seq对NLRC5结合启动子区域进行全基因组的分析,结合RNA-Seq分析可以更准确的弄清该基因是否对STAT3, MHCI以及IL18等基因存在调控作用,再通过EMSA,点突变以及敲除和过表达实验来进一步的确认NLRC5可能存在的调控机制。最终转入白痢沙门氏菌SPI-2 Ⅲ型分泌系统对巨噬细胞中MHCI有关通路的调控作用的研究,以期揭示白痢沙门氏菌隐性感染和携带状态形成的具体机制。
[Abstract]:NOD like receptor family, CARD domain containing 5 (NLR family, CARD domain containing 5, NLRC5) play an important role in the innate immune cells and immune regulation, so far, NLRC5 has been demonstrated in human and mouse can regulate the expression of MHCI molecules, and has inhibitory effects on the expression of NF- B and type I interferon pathways. Salmonella pullorum disease is one of the most important bacterial diseases in China poultry breeding, the disease outbreak caused serious economic losses to poultry production. Recessive Salmonella infection and carrier status form will lead to repeated and difficult to control. The incidence of Salmonella invasion of chicken in vivo, Class could inhibit MHC induced expression of IL10 and I, this mechanism is blocking the process of elimination resulting in one of the reasons for carrying state and persistent infection. Chicken NLRC5 molecules and Salmonella The molecular mechanism of removal. As of high-throughput studies currently infected with Salmonella after tissue level has not been reported, due to differences in other strains of pathogenic and pathological mechanism can only provide some reference, so it is necessary to systematically study the spectrum changes in the expression of Salmonella pullorum infection, affecting the expression of law as well as promoter deletion and on the basis of further study of NLRC5 in poultry and related gene expression of NLRC5, so as to analyze the possible mechanism by regulation, mechanism model of NLRC5 regulation of poultry to help establish a better understanding of the poultry Salmonella infection mechanism and the mechanism of the formation of recessive bacterial pathogen clearance. Research content.1. and the results are as follows: the first section of the second chapter, comparison of different strains of Salmonella infection leads to changes in the expression of chicken spleen and cecum by spectrum. Agilent chicken whole genome microarray, respectively for 7dpi and poly infection diarrhea, enteritis (I:C) in spleen and cecum expression spectrum analysis, simultaneous detection of IL-la, IFNy, IgG and IgM concentration, intestinal bacteria loading and immunofluorescence identification of spleen microbial load. The results showed that the pathogen triggers system Salmonella infection. The main characteristics of the immune response in Salmonella pullorum infection group, spleen expression enrichment analysis showed that the expression of differentiation and proliferation of genes mainly enriched in lymphocytes, thus showing a valid activation state of acquired immunity; one with the Thl and NK lymph cell activation related important molecules such as IL15, CD28 and B were down regulated, lymphocyte activation related genes such as IL7 increased. This indicates that the removal of cell immune function to a certain extent has been suppressed System, antibody response and B cells in the cecal tissue is activated. Infection of Salmonella group, down regulated genes mainly related to innate immunity, that natural immunity or weaken the cellular immune response activity, presumably related with protective antibodies enhanced. The second chapter second section. In that process in 7dpi cause the underlying cellular immune function weakened on Salmonella infection, and then use the time series expression analysis of 2 hours of chickens infected with Salmonella after 4 hours, 8 hours, 24 hours, 3 days, 5 days, 7 days, 12 days, 21 days were important molecular events tissue. Simultaneous determination of body weight, spleen index, bursal index, thymus index, and IL-1 alpha, IFN gamma, IgG and IgM concentration. Through the analysis of WGCNA and phenotype correlation, co expression characteristics between the probes, Wayne analysis method of mining cause latent infection The difference of gene expression and carrying. Temporal expression spectrum analysis preliminarily expression changes in spleen tissue critical period of Salmonella infection, observed natural immunity to humoral immunity is an important molecular event in the process of development and change in spleen tissue, including lymphocyte activation, proliferation and migration events; and to identify a large number of key regulatory genes, such as MHC class, I B-F, ANXA13, IL18, BF2, TNFRSF13B, BLB2 and CD28; enrichment pathway gene shows two kinds of highly negative related functional groups; differential expression and expression pattern of genes in the spleen index node highly positive correlation; in different stages of the immune response the pathway showed different characteristics with other signaling pathways and metabolic pathways; differentially expressed genes were reported in most can locate the anti body and poultry The titer of the QTL region; NLRC5 is differentially expressed genes; 3dpi-5dpi is the key stage of natural immune suppression and cell immune activation, and also is the important time to remove pathogens, gene expression in response to a trend feature chicken's immune response to pathogenic differential expression of the stage; the expression pattern of NLRC5 showed that NLRC5 translation and excitation the downstream pathway promoted scavenging reactions and pathogenic immune cells, but may also be the regulation of negative regulatory factors. The second chapter third section, and then the expression test based on the microarray data, and assuming the role of NLRC5 in regulation of the known MHCI, to carry out targeted expression of NLRC5 in the co expression data in spectrum analysis and try to find the important co expression of molecular. Analysis results showed that the co expression of NLRC5 and TAP1 and IL21R highly co expressed, as TAP1 molecules in MHCI region can be Can be NF- K B regulation but also by the effect of.2. third NLRC5 in the first chapter, in view of the importance of NLRC5 gene and its function in the process of expression and regulation function of Salmonella pullorum infection, this study further study of chicken NLRC5 gene. Firstly, cloning the full-length CDS and chicken NLRC5 the sequence of UTR, and carries on the detailed analysis of the biological information. At the organizational level of coverage may be correlated with NLRC5 fluorescence quantitative gene detection and analysis. The third chapter second section, then the fragment was constructed based on clone overexpression and RNAi vector, transfected DF1 cell line, and to build a stable expression strain. Firstly, on the basis of immunofluorescence staining, then the cells were stimulated with LPS, to observe the expression of genes related to changes in trend. Finally, expression cell line of NLRC5 and the signal pathway in the stable The expression patterns of key genes were compared and analyzed, and put forward the hypothesis of the regulatory mechanism of.NLRC5 cloning and analysis of the results of NLRC5 and the key genes showed that chicken NLRC5 and mammalian NLRC5 genes have homology, and the mammalian gene function may be inferred by entering the nucleus can play a role, and by the expression of the table key epigenetic modifications of.NLRC5 gene and its signal pathway in cell and tissue found that NF- kappa B2 and NLRC5. The co expression of STAT1 and NLRC5 was linear, the chicken has no obvious co expression characteristics, presumably the regulation of NLRC5 is complex or there may be no regulation, and STAT3 may act as a negative regulator NF- kappa B2 negative feedback regulation of the expression of NLRC5. In conclusion, in addition to MHCI, NLRC5 may regulate the expression of STAT3 and IL18. NLRC5 may have to feed poultry The mechanisms that regulate the expression of.3. in the fourth chapter, different animal milk, the specific process for the further understanding of NLRC5 regulated, and further prove that the control effect of chicken NLRC5 on MHCI, SPI-2 in type III secretion system to lay the foundation for research on latent infection and formation of bacteria carrying state and help better, this study investigated the NLRC5 start codon before the activity of different fragments of 4372bp region, and to analyze the potential regulation principle of.NLRC5 series deletion results show that NLRC5 may have 2 active promoter region, and by 617 to 1448 of the area between the isolation of two promoter activity; second promoter activity than the first promoter the activity in this area, and found that the NF- kappa B binding motif is reported and the expression and regulation of NLRC5 transcription factors closely related to the 1448 to 4372 of the fragments may be the spleen The main start organization play a role in the transcriptional expression of NLRC5 fragments, mainly from the beginning of 2108; the downstream regulation may be more complex and have a significant impact on the expression of downstream components of NLRC5; 1-2108 of the activity was only 1448-4372 of about 50%, which indicates that the activity of the 1448-2108 than the 1828 to 2108 and low activity. That 1828 to 2213 activity will be further enhanced, even more than 1448 to 4372 of the activity, so that the NLRC5 core promoter is 1828 to 2213 pieces, the interval contains the element may have STAT1, P300, NF- kappa B, MRF2, FAC1, PAX5, CRFB, CPBP, NF-AT1, MRF-2, AML1 FAC1, CRX, RelA-p65., STAT1 and NF- were in the kappa B transcription start site of 182bp before the starting position was identified. The future research should confirm the mechanism of poultry NLRC5 on regulation of MHCI, by further ChIP and ChIP-Seq binding to the promoter of NLRC5 Regional analysis of the whole genome, RNA-Seq analysis can more accurately find out whether the gene of STAT3, MHCI and IL18 are regulated by EMSA gene, point mutation and knockout and overexpression experiments to further confirm the NLRC5 regulation may exist. Regulation of the final turn to Salmonella pullorum SPI-2 type III secretion system of macrophages in the MHCI pathway, in order to reveal the latent infection of Salmonella and carrier status of the formation of the specific mechanism.

【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.4

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