本文關(guān)鍵詞：LPS誘導(dǎo)的人源IRG1基因表達(dá)譜分析及小鼠胸腺Th17細(xì)胞的分化　出處：《武漢大學(xué)》2011年博士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：研究目的：自1995年小鼠免疫應(yīng)答基因1 (Immune responsive gene 1, Irgl)首次被發(fā)現(xiàn)以來(lái),在LPM、M.avium ssp. Paratuberculosis (MAP)和促炎癥細(xì)胞因子刺激誘導(dǎo)的巨噬細(xì)胞和樹(shù)突狀細(xì)胞(Dendritic cells, DCs)中也證實(shí)了Irg1基因的特異性表達(dá)或表達(dá)升高。同時(shí),發(fā)現(xiàn)Irg1基因在妊娠早期特定階段的子宮內(nèi)腔內(nèi)皮細(xì)胞中表達(dá)迅速上調(diào),以及在弓形蟲(chóng)顱內(nèi)誘導(dǎo)的小鼠CCR9+神經(jīng)小膠質(zhì)細(xì)胞中特異性表達(dá)。這些結(jié)果表明,Irg1基因可能在宿主巨噬細(xì)胞介導(dǎo)的抵抗革蘭氏陰性菌感染的防御中或感染所致的炎癥反應(yīng)中發(fā)揮了重要作用,是一個(gè)功能未知的免疫應(yīng)答基因。 值得提出的是,人源IRG1基因的真實(shí)表達(dá)形式尚未被證實(shí),到目前為止仍處于預(yù)測(cè)階段,且其預(yù)測(cè)序列在近幾年內(nèi)一共經(jīng)歷了4次更新。EST文庫(kù)分析表明,人源IRG1基因可能在嬰兒或成人時(shí)期的生殖細(xì)胞腫瘤、白血病患者或健康人群的外周血或肺組織中表達(dá)。其功能和基因表達(dá)的調(diào)控機(jī)制尚不清楚。 另外,胸腺作為T(mén)細(xì)胞分化、增殖和成熟的主要場(chǎng)所,在胸腺細(xì)胞的陽(yáng)性選擇和陰性選擇過(guò)程中發(fā)揮了重要作用,且胸腺內(nèi)T細(xì)胞的分化發(fā)育受到胸腺微環(huán)境的嚴(yán)謹(jǐn)調(diào)控。Th1和Th2細(xì)胞是機(jī)體內(nèi)兩大主要的CD4+T細(xì)胞亞群,分別表達(dá)各自不同的細(xì)胞因子發(fā)揮不同的效應(yīng)功能,Thl7和Treg兩類(lèi)功能相對(duì)的細(xì)胞亞群的發(fā)現(xiàn)是對(duì)獲得性免疫系統(tǒng)中效應(yīng)性CD4+T細(xì)胞的一個(gè)很好的補(bǔ)充,而Thl7細(xì)胞在胸腺內(nèi)的分化發(fā)育機(jī)制尚不完全清楚。 研究?jī)?nèi)容：本研究主要檢測(cè)和證實(shí)IRG1基因在人體內(nèi)的真實(shí)表達(dá)情況,即在人體發(fā)育的何種階段(胚胎、嬰兒、幼兒或成人)、何種狀態(tài)(生理或病理)、何種組織部位(肝、脾、肺或外周血)、以何種形式(真實(shí)表達(dá)序列)存在。為了進(jìn)一步探索IRG1基因在人體中的功能及其表達(dá)調(diào)控機(jī)制,我們制備了人源IRG1蛋白抗原特異性的抗體。另一方面,我們探索了多種刺激條件下成鼠胸腺中效應(yīng)性T細(xì)胞的分化情況。 研究方法：我們分別針對(duì)不同的人源IRG1基因預(yù)測(cè)序列,設(shè)計(jì)了多對(duì)基因特異性引物;同時(shí),根據(jù)IRG1基因預(yù)測(cè)序列對(duì)應(yīng)的氨基酸序列作Blast比對(duì)分析,選取同源性較高的幾個(gè)物種,在保守區(qū)內(nèi)設(shè)計(jì)簡(jiǎn)并引物,同時(shí),選取了來(lái)源于入胚胎的多個(gè)器官組織、正常健康成人的外周血和淋巴腫瘤細(xì)胞系作為研究對(duì)象,并結(jié)合LPS刺激條件,RT-PCR檢測(cè)人體中IRG1基因的真實(shí)表達(dá)情況。通過(guò)分離克隆擴(kuò)增得到的IRG1基因片段,測(cè)序鑒定了其表達(dá)的真實(shí)性。通過(guò)大腸桿菌原核表達(dá)系統(tǒng)大量表達(dá)重組IRG1 s融合蛋白,以此作為免疫抗原免疫動(dòng)物,制備抗IRG1蛋白特異性的多克隆抗體。 另一方面,我們通過(guò)RT-PCR、ELISA和流式細(xì)胞術(shù)等方法對(duì)LPS刺激誘導(dǎo)的胸腺細(xì)胞中Th1、Th2、Th17和Treg細(xì)胞及其細(xì)胞相關(guān)的分子表達(dá)情況進(jìn)行了監(jiān)測(cè)和鑒定。 研究結(jié)果：經(jīng)過(guò)電泳檢測(cè)和測(cè)序分析,我們發(fā)現(xiàn)人源IRG1基因在胚胎和LPS刺激的健康成人PBMCs中特異性表達(dá),且在LPS刺激的成人PBMCs中的表達(dá)具有時(shí)序性。測(cè)序分析鑒定了該基因的真實(shí)表達(dá),通過(guò)構(gòu)建重組原核表達(dá)質(zhì)粒,以重組IRG1 s融合蛋白連續(xù)多次免疫動(dòng)物制備多克隆抗體,其特異性和效價(jià)均滿(mǎn)足實(shí)驗(yàn)需要。同時(shí),應(yīng)用該抗體檢測(cè)了IRG1蛋白在人體組織樣本中的表達(dá)。 另一方面,小鼠胸腺細(xì)胞經(jīng)LPS刺激誘導(dǎo)后出現(xiàn)一群特征性明顯的Th17細(xì)胞,且這群Th17細(xì)胞具有刺激物劑量效應(yīng)和時(shí)間依賴(lài)性。雖然Irg1基因在LPS刺激的胸腺細(xì)胞中表達(dá),但是與Th17細(xì)胞相關(guān)分子的表達(dá)水平不一致。 研究意義：我們首次在人體中檢測(cè)到了IRG1基因的表達(dá),且克隆到了該基因的部分片段,證實(shí)了人源IRG1基因的真實(shí)存在,并為下一步獲得全長(zhǎng)cDNA序列奠定了基礎(chǔ)。同時(shí),抗人源IRG1蛋白的多克隆抗體的制備也為進(jìn)一步探索IRG1基因在人體中的功能及其表達(dá)調(diào)控機(jī)制提供了一個(gè)有力的研究工具。 LPS刺激誘導(dǎo)的小鼠胸腺中Th17細(xì)胞分化,說(shuō)明了胸腺可以作為T(mén)h17細(xì)胞分化的場(chǎng)所,且成年小鼠的胸腺中可能存在一群有活性的Th17前體細(xì)胞,經(jīng)LPS刺激后能誘導(dǎo)向Th17細(xì)胞分化。Th17細(xì)胞分化的劑量效應(yīng)和時(shí)間依賴(lài)性表明這群細(xì)胞可能在宿主抵抗嚴(yán)重的革蘭氏陰性菌感染過(guò)程中發(fā)揮了重要作用,也為研究Th17細(xì)胞的分化發(fā)育機(jī)制提供了前提。另外,Irg1基因作為抗感染免疫應(yīng)答過(guò)程中的一個(gè)重要調(diào)控分子,并不參與調(diào)節(jié)LPS刺激誘導(dǎo)的胸腺Th17細(xì)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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392.1

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 麻莉,劉友生,王曉東,章容,李永旺;內(nèi)毒素結(jié)合肽在燒傷合并內(nèi)毒素血癥大鼠模型中的作用[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2004年05期

2 肖睿t，

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