LPS诱导的人源IRG1基因表达谱分析及小鼠胸腺Th17细胞的分化

发布时间：2017-12-31 02:22

本文关键词：LPS诱导的人源IRG1基因表达谱分析及小鼠胸腺Th17细胞的分化　出处：《武汉大学》2011年博士论文　论文类型：学位论文

【摘要】：研究目的：自1995年小鼠免疫应答基因1 (Immune responsive gene 1, Irgl)首次被发现以来,在LPM、M.avium ssp. Paratuberculosis (MAP)和促炎症细胞因子刺激诱导的巨噬细胞和树突状细胞(Dendritic cells, DCs)中也证实了Irg1基因的特异性表达或表达升高。同时,发现Irg1基因在妊娠早期特定阶段的子宫内腔内皮细胞中表达迅速上调,以及在弓形虫颅内诱导的小鼠CCR9+神经小胶质细胞中特异性表达。这些结果表明,Irg1基因可能在宿主巨噬细胞介导的抵抗革兰氏阴性菌感染的防御中或感染所致的炎症反应中发挥了重要作用,是一个功能未知的免疫应答基因。值得提出的是,人源IRG1基因的真实表达形式尚未被证实,到目前为止仍处于预测阶段,且其预测序列在近几年内一共经历了4次更新。EST文库分析表明,人源IRG1基因可能在婴儿或成人时期的生殖细胞肿瘤、白血病患者或健康人群的外周血或肺组织中表达。其功能和基因表达的调控机制尚不清楚。另外,胸腺作为T细胞分化、增殖和成熟的主要场所,在胸腺细胞的阳性选择和阴性选择过程中发挥了重要作用,且胸腺内T细胞的分化发育受到胸腺微环境的严谨调控。Th1和Th2细胞是机体内两大主要的CD4+T细胞亚群,分别表达各自不同的细胞因子发挥不同的效应功能,Thl7和Treg两类功能相对的细胞亚群的发现是对获得性免疫系统中效应性CD4+T细胞的一个很好的补充,而Thl7细胞在胸腺内的分化发育机制尚不完全清楚。研究内容：本研究主要检测和证实IRG1基因在人体内的真实表达情况,即在人体发育的何种阶段(胚胎、婴儿、幼儿或成人)、何种状态(生理或病理)、何种组织部位(肝、脾、肺或外周血)、以何种形式(真实表达序列)存在。为了进一步探索IRG1基因在人体中的功能及其表达调控机制,我们制备了人源IRG1蛋白抗原特异性的抗体。另一方面,我们探索了多种刺激条件下成鼠胸腺中效应性T细胞的分化情况。研究方法：我们分别针对不同的人源IRG1基因预测序列,设计了多对基因特异性引物;同时,根据IRG1基因预测序列对应的氨基酸序列作Blast比对分析,选取同源性较高的几个物种,在保守区内设计简并引物,同时,选取了来源于入胚胎的多个器官组织、正常健康成人的外周血和淋巴肿瘤细胞系作为研究对象,并结合LPS刺激条件,RT-PCR检测人体中IRG1基因的真实表达情况。通过分离克隆扩增得到的IRG1基因片段,测序鉴定了其表达的真实性。通过大肠杆菌原核表达系统大量表达重组IRG1 s融合蛋白,以此作为免疫抗原免疫动物,制备抗IRG1蛋白特异性的多克隆抗体。另一方面,我们通过RT-PCR、ELISA和流式细胞术等方法对LPS刺激诱导的胸腺细胞中Th1、Th2、Th17和Treg细胞及其细胞相关的分子表达情况进行了监测和鉴定。研究结果：经过电泳检测和测序分析,我们发现人源IRG1基因在胚胎和LPS刺激的健康成人PBMCs中特异性表达,且在LPS刺激的成人PBMCs中的表达具有时序性。测序分析鉴定了该基因的真实表达,通过构建重组原核表达质粒,以重组IRG1 s融合蛋白连续多次免疫动物制备多克隆抗体,其特异性和效价均满足实验需要。同时,应用该抗体检测了IRG1蛋白在人体组织样本中的表达。另一方面,小鼠胸腺细胞经LPS刺激诱导后出现一群特征性明显的Th17细胞,且这群Th17细胞具有刺激物剂量效应和时间依赖性。虽然Irg1基因在LPS刺激的胸腺细胞中表达,但是与Th17细胞相关分子的表达水平不一致。研究意义：我们首次在人体中检测到了IRG1基因的表达,且克隆到了该基因的部分片段,证实了人源IRG1基因的真实存在,并为下一步获得全长cDNA序列奠定了基础。同时,抗人源IRG1蛋白的多克隆抗体的制备也为进一步探索IRG1基因在人体中的功能及其表达调控机制提供了一个有力的研究工具。 LPS刺激诱导的小鼠胸腺中Th17细胞分化,说明了胸腺可以作为Th17细胞分化的场所,且成年小鼠的胸腺中可能存在一群有活性的Th17前体细胞,经LPS刺激后能诱导向Th17细胞分化。Th17细胞分化的剂量效应和时间依赖性表明这群细胞可能在宿主抵抗严重的革兰氏阴性菌感染过程中发挥了重要作用,也为研究Th17细胞的分化发育机制提供了前提。另外,Irg1基因作为抗感染免疫应答过程中的一个重要调控分子,并不参与调节LPS刺激诱导的胸腺Th17细胞的分化。
[Abstract]:Objective: To study the immune response of mice from 1995 1 gene (Immune responsive gene 1, Irgl), was first discovered in LPM, M.avium ssp. Paratuberculosis (MAP) and proinflammatory cytokines induced by macrophages and dendritic cells (Dendritic cells DCs) also confirmed the specificity of Irg1 gene expression or expression increased. At the same time, found to rapidly increase the expression of Irg1 gene in certain stage of early pregnancy uterine cavity in endothelial cells, and CCR9+ mouse microglia induced by Toxoplasma gondii in intracranial specific expression. These results suggest that Irg1 gene may play an important role in the inflammatory response against gram negative bacteria host macrophage mediated defense against infection or caused by infection, the immune response is a gene of unknown function.
It is noteworthy that human IRG1 gene expression has not yet been confirmed true, so far is still in the stage of prediction, and the prediction sequence in recent years has experienced a total of 4 times to update the.EST library analysis showed that human IRG1 gene in germ cell tumors in infant or adult period, or the expression of leukemia patients healthy peripheral blood or lung tissue. The expression of gene function and regulation mechanism is not clear.
In addition, the thymus as T cell differentiation, proliferation and maturation of the main places, play an important role in thymocyte positive selection and negative selection process, and the differentiation of T cells in thymus development by thymic microenvironment strict regulation of.Th1 and Th2 cells in the body are two main subsets of CD4+T cells, respectively. The expression of different cytokines exert the effects of different functions, Thl7 and Treg two found relative cell subsets is a good complement to the effect of the acquired immune system in CD4+T cells, and the differentiation of Thl7 cells in the thymus development mechanism is not entirely clear.
Research content: the purpose of this study is to detect and confirm the true expression of IRG1 gene in the human body, which is in what stage of human development (embryonic, infant, child or adult), which state (physiological or pathological), which tissues (liver, spleen, lung or peripheral blood), in what form (real expressed sequence). In order to further explore the function of IRG1 gene in the human body and its expression regulation mechanism, we prepared antigen specific antibody of IRG1 protein source was obtained. On the other hand, we explore a variety of stimulus conditions into effect in rat thymus differentiation of T cells.
Methods: we respectively according to different prediction of human IRG1 gene sequence, designed for gene specific primers for Blast analysis; at the same time, according to the amino acid sequence of IRG1 gene sequence prediction, selection of several species of high homology in the conserved region, in the design of degenerate primers. At the same time, selected from multi organs into embryos, peripheral blood of healthy adults and lymph tumor cell lines as the research object, and combined with LPS stimulation conditions, true expression of IRG1 RT-PCR gene in human detection. Through the isolation and cloning of amplified IRG1 gene fragment by sequencing, identification of the authenticity of its expression by prokaryotic. The expression of Escherichia coli expression system of recombinant IRG1 s fusion protein as antigen immune animal, preparation of anti IRG1 protein specific polyclonal antibody.
On the other hand, we monitored and identified Th1, Th2, Th17 and Treg cells and their cell related molecular expressions in thymocytes stimulated by LPS by RT-PCR, ELISA and flow cytometry.
Results: after electrophoresis detection and sequencing analysis, we found that the specific expression of human IRG1 gene in embryonic and LPS stimulation of PBMCs in healthy adults, and adults in LPS stimulated PBMCs expression in sequence. The sequencing analysis has identified the true expression of the gene, through the construction of Recombinant Prokaryotic expression plasmid, to recombinant IRG1 s fusion protein has repeatedly immunized animal polyclonal antibody, the specificity and titer both meet the need of the experiment. At the same time, the application of antibody detection the expression of IRG1 protein in human tissue samples.
On the other hand, mouse thymus cells stimulated by LPS group characteristic of Th17 cells after induction, and this group of Th17 cells with stimulus dose and time dependent manner. Although the expression of Irg1 gene in LPS stimulated thymocytes, but related to Th17 cell molecular expression was not consistent.
The significance of the study for the first time in the human body: we detected the expression of IRG1 gene, and cloned the fragment of the gene, confirmed that the human IRG1 gene exist, and for the next step to obtain the full-length cDNA sequence of the foundation. At the same time, the anti human IRG1 protein polyclonal antibody preparation to further explore the function of IRG1 gene in the human body and its expression regulation mechanism provides a powerful research tool.
Mouse thymus LPS stimulation induced Th17 cell differentiation, the thymus can be used as the differentiation of Th17 cells and adult mouse thymus sites may exist in a group activity of Th17 precursor cells after LPS stimulation can induce differentiation of Th17 cells to the dose effect of.Th17 cell differentiation and time dependence showed that these cells may play an important role in the resistance of gram negative bacteria in severe host infection process, but also provides the premise for the development of mechanisms for the differentiation of Th17 cells. In addition, the Irg1 gene as a key regulator of anti infection immune response in the process of differentiation is not involved in the regulation of LPS induced thymic Th17 cells.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

【共引文献】