细胞因子对调节性T细胞发育调节及自身免疫性骨髓纤维化发病机制研究

发布时间：2018-03-03 15:01

本文选题：转化生长因子β　切入点：白介素2　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分细胞因子对调节性T细胞的发育调节研究揭示诱导机体免疫耐受打破的因素对于理解自身免疫性疾病的发生发展有重要作用。Treg细胞是控制机体过度免疫应答阻止自身免疫性疾病发生的负调性细胞,越来越多的研究也表明Treg还参与到细菌及病毒感染、肿瘤、器官移植耐受、组织修复等多种疾病过程。因此,对Treg发育调节的研究有利于进一步认识Treg的功能,从而将其作为多种疾病的治疗靶点。Treg细胞可由胸腺发育及外周诱导两种途径产生,其发育分化受到细胞因子的调节。在CD4启动子下表达显性失活TGF-β⒏Ⅱ型受体的dnTGF-βRⅡ小鼠会出现T细胞过度活化而导致的肝脏和肠道炎性细胞浸润,其Treg细胞的抑制功能降低。Il2ra-/-小鼠由于Treg功能失调,出现多克隆T细胞和B细胞的扩增而导致多脏器自身免疫性疾病。在本研究中,我们利用Il2ra-/-Tg小鼠,探究TGF-β与IL-2信号在Treg发育过程中的共同调节作用。我们发现Il2ra-/-Tg小鼠出现类似于Treg缺陷的scurfy小鼠症状,多个脏器出现淋巴细胞浸润与病理损伤。Il2ra-/-Tg小鼠淋巴结肿大,其中T细胞明显增加,而且活化状态增强,分泌炎性细胞因子IFN-γ的能力也增强。进一步探究Il2ra-/-Tg小鼠淋巴结肿大的原因时我们发现,该小鼠Tfh细胞的比例和数量明显增加,同时生发中心B细胞也明显增多,淋巴结中浆细胞明显增多,而且非淋巴组织中和出现了浆细胞的浸润。我们发现其外周Treg比例降低而胸腺Treg比例升高。I/2ra-/-Tg小鼠Treg细胞处于活化状态,表达更高水平的Treg功能分子和活化性分子。同时,胸腺Treg体外抑制功能降低而外周Treg抑制功能正常。Il2ra-/-Tg小鼠Treg细胞高表达Th1型趋化因子受体CXCR3和转录因子Eomes,同时具有更强的IFN-γ分泌能力,TSDR区域的去甲基化水平也升高。我们发现Il2ra-/-Tg小鼠胸腺及外周Treg上Nrp-1和PD-1的表达明显降低,伴随着的是Tfr细胞的消失。利用骨髓嵌合实验补充正常的Tfr细胞能抑制Il2ra-/-Tg小鼠Tfh细胞的增加及生发中心反应的增强,说明Tfr的缺陷是小鼠淋巴结肿大及生发中心反应增强的主要原因。综上所述,我们发现了 TGF-β和IL-2信号共同作用调节了 Nrp-1+Treg细胞以及Tfr细胞的发育,为生发中心依赖的自身抗体介导的自身免疫性疾病的治疗提供了新靶点。第二部分自身免疫性骨髓纤维化的发病机制研究骨髓纤维化是一种髓系细胞增生性肿瘤,其特征包括干细胞来源的髓系细胞克隆增殖、骨髓成纤维细胞增多和网状纤维分布导致的骨髓纤维化、贫血、脾肿大、髓外造血等。通常情况下,骨髓纤维化并不被认为是一种自身免疫性疾病。但是在一些情况下,骨髓纤维化可能伴随自身免疫病出现,尤其是系统性红斑狼疮(SLE),称之为自身免疫性骨髓纤维化。自身免疫性骨髓纤维化可伴随原发性胆汁性胆管炎(PBC),但其发生机制并不清楚。我们前期发现p40-/-IL-2Rαc-/-模型小鼠是很好的PBC模型小鼠,并会自发出现肝脏的纤维化。而我们进一步发现,模型小鼠会出现脾脏肿大、贫血等现象,与临床骨髓纤维化症状相似。因此我们探究了p40-/-IL-2Rα-/-模型小鼠作为自身免疫性骨髓纤维化的模型的可能性,并进一步探究及其发病机制。不同于对照小鼠,p40-/-IL-2Rα-/-鼠脾脏出现类似CFU-S实验的结节,有大量巨核细胞,并且肝脏出现血岛。在小鼠的外周血、肝脏、脾脏中均出现了造血干细胞(LSK)样的细胞,说明髓外造血的存在。这些升高的LSK细胞与小鼠脾脏重量及肝脏的炎症程度成正相关。与对照鼠小鼠相比,血液中红细胞计数、血红蛋白、血球容积、白细胞计数均明显降低,说明出现了贫血的情况。HE染色发现p40-/-IL-2Rα-/-鼠骨髓会出现成纤维细胞的增多,银染发现骨髓会有大量网状纤维,说明小鼠出现了骨髓纤维化。骨髓中LSK样细胞增多,且造血能力降低。p40-/-IL-2Rα-/-鼠骨髓浸润大量活化状态的CD4+和CD8+ T细胞,这些T细胞有更强的IFN-y分泌能力且与LSK细胞比例呈正相关。利用转基因小鼠我们发现,敲除CD4并不会减轻小鼠的骨髓纤维化,但是敲除CD8a或者敲除IFN-γ都可以阻止骨髓纤维化的发生,表现为髓外造血消失,骨髓造血干细胞恢复正常,骨髓成纤维细胞消失以及无网状纤维分布。关键的是,在小鼠发病之后利用抗CD8α抗体清除CD8+T细胞,能显著减轻小鼠自身免疫性骨髓纤维化的发生。总之,我们发现CD8+ T细胞及其产生的细胞因子IFN-γ是导致小鼠自身免疫性骨髓纤维化的关键因素。利用抗体清除CD8+ T细胞能对自身免疫性骨髓纤维化进行治疗,为临床上治疗提供新的思路。
[Abstract]:The first part studies revealed the cytokines regulate the immune tolerance induced by the factors for the occurrence and development of breaking the understanding of autoimmune diseases have an important role in.Treg cell is negative control cell body immune oerreaction tonality to prevent autoimmune disease of regulatory T cell development, more and more studies show that Treg is also involved in bacteria and virus infection, tumor, organ transplantation tolerance, tissue repair and other diseases. Therefore, is conducive to further understanding of Treg function on Treg development regulation, so it can be used as a therapeutic target in.Treg cells of various diseases can be caused by the development of thymus and peripheral in two ways, the differentiation and development by regulating cell the dominant factor. The inactivation of the TGF- beta type II receptor dnTGF- beta R II in mice caused by excessive activation of T cells in the liver and the expression of CD4 promoter Intestinal inflammatory cell infiltration, inhibit the function of Treg cells in.Il2ra-/- mice reduced disorders due to the Treg function, the amplification of polyclonal T cells and B cells and cause multiple organ autoimmune diseases. In this study, we used Il2ra-/-Tg mice, regulated by exploring the role of TGF- beta and IL-2 signal in Treg development process. We found that Il2ra-/-Tg mice showed symptoms similar to scurfy mice deficient in Treg, multiple organs appeared lymphocytic infiltration and pathological enlargement of lymph nodes of mice.Il2ra-/-Tg, T cells were significantly increased, and the activation state of enhanced secretion of inflammatory cytokines IFN- gamma is also enhanced. To further explore the causes of lymphadenopathy in Il2ra-/-Tg mice we found that the proportion of Tfh cells in mice and significantly increased the number at the same time, the germinal center B cells also increased significantly, the lymph node of plasma cells increased significantly, and And non lymphoid tissue and the infiltration of plasma cells. We found that Treg decreased the proportion of peripheral and thymus Treg increased.I/2ra-/-Tg activation in mouse Treg cells, the expression of Treg molecules and higher levels of activation of molecules. At the same time, the thymus Treg in vitro inhibition function decreased peripheral Treg inhibited expression of Th1 chemokines CXCR3 receptor and transcription factor Eomes of Treg cells in mice with normal.Il2ra-/-Tg function, IFN- also has a stronger gamma secretion capacity, TSDR region demethylation level also increased. We found that the expression of Nrp-1 and PD-1 and Il2ra-/-Tg week Treg mice thymus was significantly decreased, with the disappearance of Tfr cells. The bone marrow chimeric experiments normal Tfr cells can inhibit the Il2ra-/-Tg of mouse Tfh cells increased and the germinal center reaction enhancement, Tfr defects is the lymph nodes and in mice The main reason was enhanced. In summary, we found that the interaction of TGF- beta and IL-2 signal regulated Nrp-1+Treg cell and Tfr cell development, provides a new therapeutic target for autoantibody induced germinal center dependent mediated autoimmune diseases. The second part of the pathogenesis of autoimmune bone marrow fibrosis fibrosis is a myeloid proliferative tumor, characterized by clonal proliferation of myeloid cells from stem cells, bone marrow fibroblasts increased and reticular fiber distribution leads to bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis. Typically, bone marrow fibrosis is not considered an autoimmune disease but in some cases, bone marrow fibrosis may be associated with autoimmune diseases, especially systemic lupus erythematosus (SLE), called autoimmune bone marrow fibrosis itself. Autoimmune myelofibrosis with primary biliary cholangitis (PBC), but its pathogenesis is not clear. We previously found that the p40-/-IL-2R alpha c-/- mouse model is a good model of PBC mice, and spontaneously liver fibrosis. And we further found that the mouse model will appear splenomegaly, anemia and other phenomena. Similar to the clinical symptoms of bone marrow fibrosis. So we study the p40-/-IL-2R - / - mice alpha possibility as autoimmune bone marrow fibrosis model, and further explore its pathogenesis. Different from control mice, spleen p40-/-IL-2R alpha - rat CFU-S like experimental nodules, there are a large number of megakaryocytes, and liver blood liver island. In the peripheral blood of mice, spleen occurred in hematopoietic stem cells (LSK) like cells, indicating extramedullary hematopoiesis. These elevated LSK cells and spleen weight in mice The degree of inflammation and liver are positively correlated. Compared with control mice, red blood cell count, blood hemoglobin, hematocrit and leukocyte count were significantly decreased, indicating the anemia.HE staining indicated that p40-/-IL-2R alpha - there will be increased into bone marrow fibroblasts, silver staining showed that bone marrow may have a large network fiber, that mice showed bone marrow fibrosis. LSK like cells in the bone marrow of.P40-/-IL-2R rats was increased, a large bone marrow infiltration of the activation state of CD4+ and CD8+ in T cells decreased and hematopoietic ability, these T cells have stronger IFN-y secretion ability and positively related with the percentage of LSK cells in transgenic mice. We found that knockdown of CD4 and do not reduce the mice bone marrow fibrosis, but CD8a knockdown or knockout of IFN- gamma can prevent bone marrow fibrosis, manifested as extramedullary hematopoiesis disappeared, bone marrow hematopoietic stem cells to restore normal bone. Pulp fibroblast cells disappeared and no reticular fiber distribution. The key is that the use of anti CD8 antibody depletion of CD8+T cells in mice after onset, can significantly reduce the mice autoimmune bone marrow fibrosis. In short, we found that the cytokines IFN- CD8+ T cells and the key factors leading to autoimmune myelofibrosis the use of antibodies to remove CD8+ T. Cells can treat autoimmune myelofibrosis, to provide new ideas for clinical treatment.

【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】：2017
【分类号】：R593.2

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