原发性胆汁性胆管炎模型鼠腹腔B细胞调节性功能机制探究

发布时间：2017-12-28 12:23

本文关键词：原发性胆汁性胆管炎模型鼠腹腔B细胞调节性功能机制探究　出处：《中国科学技术大学》2016年博士论文　论文类型：学位论文

【摘要】：原发性胆汁性胆管炎(PBC)是一种自身免疫性肝脏疾病,它的典型特征是肝脏有大量淋巴细胞浸润在胆管周围,并且血清中有高滴度的由自身反应性B细胞分泌的抗线粒体抗体AMAs。有很多因素参与到PBC的发病过程中,包括免疫失调、遗传的易感性以及环境因素等。已有的研究指出B细胞在PBC的发病过程中的作用十分复杂,根据情况不同,可能发挥致病性作用或者免疫调节性功能。但是其具体机制尚不清楚,仍然需要进一步研究和探索。B细胞是一类重要的免疫细胞,主要分为2个亚群,即B1细胞(包含B1a细胞和Blb细胞)和B2细胞。这两个B细胞亚群在前体细胞来源、发育途径、组织分布、表型及功能等方面存在差异。在自身免疫性疾病发生过程中,一般认为B细胞通过分泌抗体发挥致病性作用。但最近的研究发现B细胞除了包含发挥致病性功能的亚群,同时还具有免疫调节性功能的亚群。调节性B细胞(Breg细胞)发挥功能的机制主要包括产生IL-10和TGF-β、参与次级抗原递呈、与致病性T细胞相互作用以及诱导调节性T细胞(Treg细胞)的产生等。B1细胞是一个特殊的B细胞亚群,大量存在于腹腔及胸腔中,主要通过分泌天然抗体、作为抗原递呈细胞以及分泌细胞因子来发挥免疫保护作用。B1细胞来源的天然抗体在细菌和病毒感染过程中发挥重要的保护作用。Bla细胞是腹腔B1细胞中的主要亚群,Bla细胞经典的功能包括产生血清中静息状态的IgM、分泌肠道IgA以及发挥免疫调节功能等。B1a细胞是抗炎性细胞因子IL-10的主要来源,并且在接触性超敏反应模型以及病理条件下能够发挥免疫调节性功能。为了更好的阐明B细胞亚群在PBC发病进程中的作用,我们以p40-/-IL-2Rα-/-小鼠以及被广泛研究的dnTGF-βRII小鼠作为PBC的模型鼠展开研究。本论文主要探究了p40-/-IL-2Rα-/-小鼠中腹腔B1a细胞的紊乱与自身免疫性胆管炎的关系,同时利用dnTGF-βRⅡ小鼠腹腔B细胞的共转输实验对模型鼠中腹腔B细胞的免疫调节性功能进行验证。目前取得的结果如下：1.p40-/-IL-2R α-/-模型鼠腹腔徽环境改变影响腹腔B细胞数目并与肝脏炎症具有相关性为了探究p40-/-IL-2Rα-/-模型鼠肝脏损伤时腹腔微环境的变化,通过检测腹腔中各细胞亚群数量,我们发现模型鼠中腹腔总细胞数目显著增多,且增多的主要是T细胞,尤其是致病性的CD8+T细胞,而B细胞数目显著减少。进一步研究表明模型鼠腹腔CD4+和CD8+T细胞分泌Thl型炎性细胞因子IFN-γ的能力显著增强。通过检测腹腔灌洗液中炎性细胞因子的水平,我们发现促炎性细胞因子TNF和MCP-1水平显著升高,这可能是导致腹腔各细胞亚群改变的原因。通过相关性分析,发现模型鼠中腹腔总细胞数与肝脏单个核细胞数呈正相关,腹腔中B1a细胞占B细胞的比例与腹腔及肝脏单个核细胞数呈负相关,且腹腔中Bla细胞占B细胞的比例与腹腔及肝脏中致病性的CD8T细胞的比例呈负相关。2. p40-/-IL-2Rα-/-模型鼠腹腔Bla细胞数目减少且功能性分子改变通过对PBC模型鼠发病过程中腹腔B细胞的详细检测,我们发现相比于对照鼠,模型鼠腹腔B1a及B1b细胞的比例和数目显著降低,而且模型鼠腹腔Bla细胞的比例随着年龄增长逐渐降低。进一步的机制研究表明模型鼠腹腔Bla细胞增殖能力显著降低且凋亡水平显著升高。模型鼠腹腔B1a细胞数目显著减少的同时,与Bla细胞免疫调节性功能相关分子,包括IL-10, CTLA-4和GITR的表达水平都显著下降；与B1a细胞活化及免疫共刺激相关分子CD44和CD80的表达水平明显降低。通过对另一种PBC模型鼠,即dnTGF-βRII小鼠的表型检测,我们发现与p40-/-IL-2Ra-/-模型鼠类似, dnTGF-βRII小鼠腹腔B1a细胞比例下降,且表达IL-10、CTLA-4、CD44和CD80的水平同样显著低于同窝对照鼠。3. p40-/-IL-2Rα-/-模型鼠腹腔Bla细胞转录组学分析为了全面探究p40-/-IL-2Ra-/-模型鼠腹腔B1a细胞转录组变化及其内在机制,利用基因芯片技术并配合荧光定量PCR验证,我们发现与对照鼠相比,模型鼠腹腔Bla细胞有381个基因发生了显著变化(2倍以上差异),其中包含大量细胞因子及其受体相互作用相关、转录因子活化相关及细胞周期相关基因的变化。且多个与B细胞功能相关的基因都发生了显著改变,其中具有免疫抑制性基序(ITIM)的分子CD72 (Cd72)、CD22 (Cd22)等表达显著上升,与流式结果一致的Cd44、Ctla4等表达显著下降,以及IL-10的上游抑制分子AHR (Ahr)等表达显著上升。这些发现详细地展示了模型鼠与对照鼠腹腔Bla细胞存在的差异,揭示了B1a细胞异常在PBC发病过程中的重要作用机制,并提示了一些在人类PBC研究中值得参考的基因和信号通路。4.PBC模型鼠腹腔B细胞对自身免疫性胆管炎治疗潜力的评估为了探讨PBC模型鼠腹腔B细胞对胆管炎是否具有治疗潜能,我们进一步利用过继转输实验发现单转输dnTGF-βRⅡ模型鼠脾脏CD8+T细胞能引起RAGI-/-受体鼠肝脏产生严重的淋巴细胞浸润,而将dnTGF-βRⅡ模型鼠腹腔B细胞或WT鼠腹腔B细胞与dnTGF-βⅠ RⅡ模型鼠脾脏CD8+T细胞共转输,发现RAG1-/-受体鼠肝脏淋巴细胞浸润都有所减轻。该实验为证明PBC模型鼠腹腔B细胞在炎性条件下具有强大的免疫调节功能提供了有利的证据。综上所述,我们发现在炎性环境下,p40-/-IL-2Rα-/-模型鼠中腹腔B1a细胞显著减少,且B1a细胞免疫调节性功能紊乱,分泌免疫调节性分子IL-10和表达CTLA-4的水平显著降低,导致腹腔B1a细胞对致病性T细胞的抑制能力下降。dnTGF-βRⅡ模型鼠腹腔Bla细胞的表型与p40-/-IL-2Rα-/-模型鼠类似。因此,我们认为PBC模型鼠腹腔B1a细胞免疫调节性功能损伤导致模型鼠病情加重,这可能为PBC的治疗提供了一个新的方向。
[Abstract]:Primary biliary cholangitis (PBC) is an autoimmune liver disease. Its typical feature is a large number of lymphocytes infiltrating around the bile duct in the liver. There is a high titer of anti mitochondrial antibody AMAs secreted by autoreactive B cells in the serum. There are many factors involved in the pathogenesis of PBC, including immune disorders, genetic susceptibility, and environmental factors. Previous studies have pointed out that the role of B cells in the pathogenesis of PBC is very complicated. Depending on the situation, it may play a pathogenic role or an immunomodulatory function. However, its specific mechanism is still unclear, and further research and exploration are still needed. B cells are an important class of immune cells, which are divided into 2 subgroups, that is, B1 cells (including B1a cells and Blb cells) and B2 cells. The two B cell subsets differ in the origin, development pathway, tissue distribution, phenotype and function of the precursor cells. In the process of autoimmune diseases, it is generally believed that B cells play a pathogenicity role by secreting antibodies. However, recent studies have found that B cells contain subsets of pathogenic functions and a subgroup of immunoregulatory functions. Regulatory B cells (Breg cells) play the functional mechanisms, mainly including the production of IL-10 and TGF- beta, participation in secondary antigen presentation, interaction with pathogenic T cells, and induction of regulatory T cells (Treg cells). B1 cell is a special B cell subgroup, which exists in the abdominal cavity and thoracic cavity. It mainly plays the role of immune protection by secreting natural antibodies, antigen presenting cells and secreting cytokines. Natural antibodies from B1 cells play an important role in the process of bacterial and viral infection. Bla cells are the main subsets of B1 cells in peritoneal cavity. The classical functions of Bla cells include the production of IgM in resting state, secretion of intestinal IgA and immune regulation function. B1a cells are the main source of anti-inflammatory cytokine IL-10, and can play an immunoregulatory function under the contact hypersensitivity model and pathological conditions. In order to better clarify the role of B cells in the pathogenesis of PBC in the process of subsets, we used p40-/-IL-2R and alpha - / - mice are widely studied dnTGF- beta RII mice as a model of PBC rats. This paper mainly explores the relationship between peritoneal B1a cells p40-/-IL-2R alpha - / - mice in disorder and autoimmune cholangitis, while the use of dnTGF- beta R II mouse peritoneal B cells were transferred immune experiments on peritoneal B cells in rat model of regulatory function verification. The results are as follows: 1.p40-/-IL-2R alpha - rat model of abdominal Hui environmental changes affecting the number of B cells and peritoneal and liver inflammation associated with peritoneal microenvironment in order to explore the changes of p40-/-IL-2R alpha - rat model of liver injury, by detecting the peritoneal cell subsets number, we found that the total number of cells in a rat model of abdominal cavity increased significantly the increase is mainly, and T cells, especially pathogenic CD8+T cells, and the number of B cells decreased significantly. Further study showed that the ability of CD4+ and CD8+T cells to secrete type Thl inflammatory cytokine IFN- gamma in the rat peritoneal cavity was significantly enhanced. By detecting the levels of inflammatory cytokines in peritoneal lavage fluid, we found that the levels of proinflammatory cytokines TNF and MCP-1 increased significantly, which may be the reason leading to the change of peritoneal cell subsets. Through correlation analysis, found that positive peritoneal mouse models of liver cells and total number of mononuclear cells, B1a cells accounted for the proportion of B cells was negatively correlated with the number of mononuclear cells of liver and abdominal abdominal cavity, abdominal cavity and Bla cells for pathogenic CD8T cells B cells and the proportion of abdominal cavity and in the liver the ratio was negatively correlated. 2. p40-/-IL-2R alpha - rat model of peritoneal Bla cells decreased in number and function of molecular changes through detailed detection of peritoneal B cells in the pathogenesis of PBC model rats, we found that compared to control mice, rat model of peritoneal B1a and B1b cells and the ratio of the number decreased significantly, but the proportion of peritoneal Bla cells in rat model with increasing age reduce. Further mechanism studies showed that the proliferation of Bla cells in the peritoneal cavity was significantly reduced and the level of apoptosis was significantly increased. The number of B1a cells in the abdominal cavity of model rats was significantly reduced, while the expression levels of Bla, including IL-10, CTLA-4 and GITR, were significantly decreased. The expression levels of CD44 and CD80 correlated with B1a cell activation and costimulatory molecules were significantly decreased. Through the phenotypic detection of another PBC model mouse, dnTGF- beta RII, we found that compared with p40-/-IL-2Ra-/- model mice, the proportion of peritoneal B1a cells in dnTGF- beta RII mice decreased, and the levels of IL-10, CTLA-4, CD44 and CD80 were also significantly lower than those in the same control mice. 3. p40-/-IL-2R alpha - / - SD rats Bla cell transcriptome analysis in order to explore the changes of p40-/-IL-2Ra-/- in rat model of peritoneal B1a cell transcriptome and its intrinsic mechanism using gene chip technology and fluorescence quantitative PCR test, we found that compared with the control rats, rats peritoneal Bla cells have undergone significant changes in 381 genes (more than 2 times the difference), which contains a large number of cell factor and its receptor interactions, changes related to activation of transcription factors and cell cycle related genes. And related to B cell function genes have undergone significant changes, which has immune inhibitory motifs (ITIM) molecular CD72 (Cd72), CD22 (Cd22) expression was significantly increased, and the flow cytometry results consistent with Cd44 and Ctla4 expression decreased significantly, and the IL-10 upstream inhibitor AHR (Ahr) the expression increased significantly. These findings show the difference between the Bla cells in the abdominal cavity of the model mice and the control mice, revealing the important mechanism of B1a cell abnormality in the pathogenesis of PBC, and suggesting that some of them are in humans.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575.7;R-332

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