【作者】 白鹰；

【导师】 唐一源；

【作者基本信息】 大连理工大学 ， 生物医学工程， 2009， 博士

【摘要】 重症肌无力（myasthenia gravis,MG）和它的动物模型实验性自身免疫性重症肌无力（experimental autoimmune myasthenia gravis,EAMG）均为自身抗体介导的神经肌肉接头（neuromuscular iunction,NMJ）疾病,NMJ突触后膜烟碱型乙酰胆碱受体（acetylcholine receptor,AChR）是大多数病人自身抗体攻击的主要靶点。MG和EAMG的这些抗体的产生依赖于辅助T细胞（helper T cell,Th）和它们产生的细胞因子。Th17为一种新定义的Th类细胞,与自身免疫性疾病关系密切,为了明确Th17细胞在MG中的作用,本研究以EAMG模型和病人胸腺瘤组织为研究对象,采用免疫学和分子生物学技术,研究Th17细胞在MG发病中的作用,结果显示:自身反应性Th17细胞具有驱使EAMG致病性抗体产生的作用,并且MG病人的胸腺瘤组织高表达IL-17。本研究提示Th17细胞是导致MG的关键因素,可以作为免疫治疗的新靶点。初始T细胞分化成Th亚群需要固有免疫和适应性免疫系统相互作用,因此本研究首先探讨了单核细胞趋化蛋白-1（monocyte chemoattractantprotein 1,MCP-1,也称CC chemokine ligand 2,CCL2）在EAMG发病机制中的作用。CCL2在C57BL/6（B6）小鼠的脾和淋巴结表达增高,CCL2缺乏(CCL2^-/-)小鼠抵抗EAMG诱导,CCL2^-/-小鼠血清抗AChR IgG、IgGl和IgG2a抗体水平与B6小鼠相似,但IgG2b水平明显减少,提示IgG2b水平低下是CCL2^-/-小鼠能抵抗EAMG诱导的原因。CCL2^-/-小鼠B细胞在体外增殖、分化成浆细胞、产生IgG抗体和迁移到次级淋巴器官的功能均正常,分泌IFN-γ（Th1）和IL-4（Th2）的CD4⁺T细胞数量略少于B6鼠,但是分泌IL-17的CD4⁺T细胞即Th17细胞却明显少于B6鼠,表明CCL2^-/-小鼠IgG2b水平的低下与Th17细胞数量不足有关。因此CCL2在EAMG发病中的作用是驱使Th17细胞的发生,之后Th17细胞辅助B细胞产生致病性IgG2b抗体介导NMJ传导功能阻滞,形成EAMG。继之的研究表明缺乏CCL2导致产生IL-6的CD11b⁺单核细胞归巢障碍,从而影响AChR反应性Th17细胞的分化,揭示了CCL2驱使Th17发生的机制是通过聚集CD11b⁺细胞归巢到次级淋巴器官产生IL-6来促使Th17细胞分化。IL-6缺乏(IL-6^-/-)小鼠抵抗EAMG的诱导缘于体内Th17细胞分化障碍,说明IL-6促使体内Th17细胞的分化来介导EAMG发病。通过给EAMG小鼠直接注射IL-17进一步探讨Th17在MG发病机制中的作用。外源性IL-17可致B6小鼠EAMG特征加重并且抗AChR特异性IgG2b抗体水平明显升高。给IFN-γ缺乏(IFN-γ^-/-)小鼠注射IL-17,可以诱导较严重的EAMG症状,说明是IL-17而不是IFN-γ更影响EAMG的形成,Th17细胞促进EAMG形成的作用不依赖于Th1细胞。对AChR有免疫性的供体小鼠的Th17细胞可介导受体重组活化基因1（recombinant active gene 1,RAG1）敲除(RAG1^-/-)小鼠形成EAMG,IL-17缺乏(IL-17^-/-)小鼠抵抗EAMG的诱导,进一步显示了Th17细胞在MG发病中的作用。为了检验Th17细胞在胸腺瘤合并MG发病机制中的作用,对行胸腺切除手术的27例胸腺瘤患者的相关资料进行了回顾分析,并将切除的胸腺瘤组织蜡块标本切片用免疫组化方法确定MCP-1、IL-17、IL-6细胞因子及CD11b⁺和CD4⁺细胞在胸腺瘤组织中的表达情况。结果显示越年轻的男性胸腺瘤患者越容易伴发MG,B1和B2型为主的胸腺瘤合并MG的可能性较大,而C型（恶性肿瘤）合并MG的可能性极小;Ⅱ型病理多表现为B1和B2型,越是良性的胸腺瘤越易发生MG,淋巴细胞型胸腺瘤易伴发MG;胸腺瘤合并MG的胸腺瘤组织高表达CCL2、IL-6和IL-17细胞因子及CD11b⁺细胞和CD4⁺细胞。总之,本研究可以得出以下结论:在EAMG发病机制中CCL2介导CD11b⁺单核细胞归巢次级淋巴器官,产生细胞源性的IL-6促进自身反应性Th17细胞分化,之后Th17细胞作用于B细胞产生能与补体结合的高致病性抗AChR IgG2b抗体,导致NMJ传导障碍,形成EAMG。CCL2在调整产生IL-6的CD11b⁺细胞、Th17细胞和B细胞之间相互影响中起重要作用,这些交互作用在自身抗体的形成和继之发展成EAMG是非常关键的,而且Th17辅助B细胞产生自身抗体不依赖于Th1。在胸腺瘤合并MG病人的研究中验证了这个机制,说明Th17细胞和其细胞因子IL-17在胸腺瘤合并MG的发病中有一定的作用,可能也会启动MG的自身免疫反应。更多还原

【Abstract】 Myasthenia gravis（MG） and its animal model experimental autoimmune myasthenia gravis（EAMG） are autoantibodies mediated diseases of neuromuscular junctions.The nicotinic Acetylcholine receptors（AChR） at the postsynaptic membrane of neuromuscular junction are primary target for autoantibody attack in the majority of MG patients.Production of these antibodies in MG and EAMG are dependent of the cytokines produced by T helper （Th） cells.In order to understand the newly defined Th-17 cells in MG,we have used EAMG model as well as thymoma tissues from MG patients.Cellular immunology and genetic approaches in the thesis work have been combined.Autoreactive Th-17 cells drives pathogenic antibody production in EAMG have been identified.Further,IL-17 is highly expressed in thymoma tissues from patients with MG.Overal,these results reveal that IL-17 as a critical player for manifestation of muscular weakness and may serve as a target for novel immuno-therapy.Since differentiation of na（?）ve T cells into Th subtype requires interactions between innate and adaptive immune system prior to T cell activation.We first examined a chemokine CCL2（CC chemokine ligand 2,also named monocyte chemoattractantprotein 1,MCP-1） in development of EAMG.CCL2 expression was elevated in C57BL/6（B6） mice in lymph nodes and in spleen.CCL2-deficient(CCL2^-/-) mice is resistant to the development of EAMG. The level of serum anti AChR IgG,IgG1 and IgG2a in CCL2^-/-mice is similar to that of B6 mice,but IgG2b level is significantly reduced,indicating the possible reason of the resistance of CCL2^-/- mice to the development of EAMG is the reduced IgG2b level.CCL2 deficiency does not impair the capacity of B cells to proliferate,differentiate into plasma cells,release IgG antibodies and migration to the secondary lymphoid organs,the number of CD4⁺T cells that secretes IFN-γ（Th1 cell） and IL-4（Th2 cell） in CCL2 mice is slightly less than that of B6 mice,but the number of CD4⁺T cells that secretes IL-17 cells is significantly reduced in CCL2^-/- mice than that of B6 mice,implicating that reduced express of IgG2b in CCL2 mice is related with the reduced Th17 cells,implicating that reduced express of IgG2b in CCL2 mice is related with the reduced Th17 cells.The results show the role of CCL2 in the development fo EAMG,that is CCL2 drives Th17 cells priming,which assist B cells to produce pathological anit-AChR IgG2b,thus induce the functional blockage of neuromuscular junction（NMJ） and cause EAMG.Subsequent investigation reveals that lack of CCL2 lead impared homing of IL-6 producing CD11b⁺ monocytes.Subsequently,differentiation of AChR-reactive Th17 cells compromised.Thus,The results revealed CCL2 recruited IL-6 producing monocyte drive Th17 cells.The resistance to EAMG in IL-6^-/- mice could derive from faulty development of Th17 cells secondary to IL-6 deficiency.This result indicates a role of IL-6 in the development of AChR-specific Th17 cells in vivo,which induces the development of EAMG.The role of IL-17 in the development of EAMG was further examined by direct injecting of IL-17 to EAMG mice.Exogenous administration of IL-17 to B6 mice EAMG model showed severe signs and specific anti AChR IgG2b level was marked elevated,as indicated by a lack of disease induction in IL-17-deficient mice.Treatment of IFN-γ^-/- mice with IL-17 eaqully exacerbate EAMG,indicating that the role for IL-17 is independent of IFN-gamma. The RAG1^-/- recipient mice transferred AChR-specific Th17 cells from B6 mice developed EAMG suggests the role of Th17 cells in the development of EAMG as indicated by a lack of disease induction in IL-17 deficient mice.Given the associations of thymoma with MG,it was next exmianed whether IL-17 is present in thymoma.A retrospective analysis of 27 cases of thymoma for whom thymectomy were performed.The immunohistochemical staining with antibodies to CCL2,IL-17,IL-6, CD11b⁺ and CD4⁺ was performed on the parafine-embedded sections of the 27 thymoma specimen.Younger patients and male patients are easy to be complicated with MG.B1 and B2 type thymoma were more likely to accompanied with MG,while the C-type（malignant tumor） has a very small possibility to do that.The more benign thymoma was,the easier MG occurred,and the possibility was high for lymphocytes thymoma to be accompanied by MG. CCL2,IL-6 and IL-17 cytokines,CD11b⁺ cells and CD4⁺ cells were over-expressed in thymoma tumor tissues in patients with MG accompanied by thymoma.Above all,this paper demonstrated that Inflammation and/or immunization recruits CD11b⁺ mononuclear phagocytes to lymph nodes,a phenomenon that depends on CCL2.In the lymph nodes,those antigen presenting cells present the autoantigen to Th0 cells and make IL-6,which polarizes the Th0 cells toward a Th17 phenotype.The Th17 cells are then required as helpers for the production of high titer of pathogenic anti-AChR IgG2b antibodies from B cells.Those antibodies are the ultimate responsible for the block at the neuromuscular junction that leads to muscular weakness.The role of Th17 cells in the development of EAMG is not dependent on Th1 cell.We show that CCL2 can facilitate the cognate interactions among IL-6-producing CD11b⁺ cells,autoreactive Th17 cells,and B cells.These interactions are critical in the genesis of autoantibodies and in the subsequent development of manifestations of diease.The evidence of that CCL2,IL-6 and IL-17 cytokines,CD11b⁺ cells and CD4⁺ cells were over-expressed in thymoma tumor tissues in patients with MG accompanied by thymoma.In the tumor tissues of thymoma accompanied with MG supports our prediction:Th17 cells have a role in B cell-mediated autoimmune diseases.In the tumor tissues of thymoma accompanied with MG,highly expressed IL-17 may also initiate the MG’s autoimmune reactions.更多还原