【作者】 杨欢；

【导师】 钮晓音；

【作者基本信息】 上海交通大学 ， 基础医学， 2016， 硕士

【摘要】 类风湿关节炎（RA）是一种慢性系统性自身免疫病;有丝分裂原活化蛋白激酶激酶激酶2（MEKK2）是MAPK信号通路中MAP3K成员之一,可参与T细胞及多种细胞因子的信号调节,其在RA发生发展中的作用暂时未知。本文将探讨MEKK2在类风湿关节炎中的作用及其机制。我们首先检测RA患者和健康对照者（HC）的外周血单个核细胞（PBMC）中MEKK2蛋白及基因表达水平,并进一步研究高度活动期RA患者经药物艾拉莫德治疗后,临床症状的变化,以及其PBMC中MEKK2蛋白及基因表达变化,探讨治疗药物艾拉莫德是否通过MEKK2发挥治疗作用,最后观察MEKK2在类风湿性关节炎患者滑膜的表达情况。实验结果显示,MEKK2在RA患者中呈低表达,而且随着RA患者病情的缓解,MEKK2表达有所上升;同时艾拉莫德药物对RA的缓解极有可能是通过对MEKK2蛋白的调节起到治疗作用。胶原诱导性关节炎（CIA）动物模型被广泛应用于RA的发病机制与治疗方法的研究,我们进一步应用Mekk2^-/-和WT小鼠,诱导CIA动物模型。在二次免疫后,观察小鼠疾病进程和临床评分,检测脾脏及引流淋巴结中辅助性T淋巴细胞（Th）亚群Th1、Th17、Tfh、Treg的比例变化及相关转录因子表达水平,并进一步检测血清中相关细胞因子的含量以及基因水平。与对照组相比,Mekk2^-/-小鼠CIA发病程度较WT小鼠严重,CIA模型组小鼠脾脏和引流淋巴结中,Th1、Th17、Tfh细胞比例均明显上升,Treg细胞比例下降;脾脏和淋巴结Th细胞相应转录因子有相同变化趋势;在CIA模型组,特别是Mekk2^-/-小鼠血清中,促炎性细胞因子IL-17、IL-21、IL-6、TNF-α等蛋白表达水平有所上升,抑制炎性细胞因子IL-10表达下降。与对照组小鼠相比,CIA模型组p38与JNK的磷酸化水平都有所升高,但CIA模型组内,Mekk2^-/-与WT小鼠的p38与JNK的磷酸化水平无显著差异。研究结果提示,MEKK2蛋白缺失引起Th细胞失衡,导致CIA发病加重。综上所述,本研究通过MEKK2在RA中的表达情况以及其与疾病严重程度的关系,并进一步揭示在CIA模型中,Mekk2^-/-小鼠的疾病情况和体内Th细胞亚群、相关细胞因子、下游通路分子的变化,发现MEKK2的表达与RA严重程度呈负相关,其缺失会引起体内Th细胞数量和功能的失衡,为类风湿性关节炎治疗寻找新靶点提供新的思路以及实验依据。更多还原

【Abstract】 Rheumatoid arthritis（RA）is a chronic autoimmune disease.Mitogen activated protein kinase kinase kinase 2（MEKK2）,a member of the MAPK signaling pathway of MAP3K,participates in regulating the signaling pathways of T cells and cytokines.However,its role in the pathogenesis of RA remains unknown.In this study,we will explore the role and mechanism of MEKK2 in RA.Firstly,we checked MEKK2 expression in peripheral blood mononuclear cells（PBMC）of RA patients and healthy controls（HC）both at protein level and gene level.Then,we examined MEKK2 expression in different phases of the disease in RA patients after Iguratimod treatment.We also compared the MEKK2 expression in the synovial tissue of RA patients and osteoarthritis（OA）patients.Our results show that MEKK2expression decreased in RA patients,and with the remission of the disease,MEKK2expression increased.In addition,after iguratimod treatment,the expression of MEKK2was up-regulated in RA patients with significant clinical improvements.We further used the animal model of RA,collagen induced arthritis（CIA）,to study the role and mechanism of MEKK2 in CIA.Mekk2^-/-and WT mice were randomly divided into CIA group and control group.After the second immunization,we observed disease progression as well as the changes of Th subsets and cytokine expressions.We found that Mekk2^-/-mice developed more severe disease.In CIA group,the effector Th cells including Th1,Th17 and Tfh were increased in Mekk2^-/-mice not only in spleen but also in draining lymph nodes,while Treg was decreased.All the Th subsets related transcriptional factors were changed accordingly.The proinflammatory cytokines,IL-17、IL-21、IL-6 and TNF-α,etc,were up-regulated in the serum of Mekk2^-/-CIA mice,and anti-inflammatory cytokine,IL-10 was down-regulated.Compared with control group,p-p38 and p-JNK was increased,but there is no significant difference in Mekk2^-/-mice and WT mice.Our results indicated that lack of MEKK2 caused the imbalance of Th subsets,leading to more severe CIA.In summary,we focused on the MEKK2 expression in RA and its relation to the disease progress,and further studied the role and mechanism of MEKK2 in CIA using Mekk2^-/-mice.Our study revealed that MEKK2 expression has negative correlation with severity of RA.Lack of MEKK2 will break the balance of Th subsets both in their quantities and their functions.Our study provide a new strategy and experimental evidences for RA therapy.更多还原