滤泡辅助性T细胞（Tfh）与自身免疫性疱病的相关性研究

  本文关键词：滤泡辅助性T细胞（Tfh）与自身免疫性疱病的相关性研究，由笔耕文化传播整理发布。

        自身免疫性疱病（autoimmune bullous diseases, ABD）是一类临床常见的自身免疫性疾病，临床特征为皮肤红斑、水疱、大疱、糜烂，多发于中老年人，病情顽固且易反复发作。ABD包括两大类：表皮内疱病和表皮下疱病，前者以天疱疮（pemphigus）最为多见，后者则以大疱性类天疱疮（bullouspemphigoid，BP）为典型代表[1]。目前由致病性自身抗体介导的疱病免疫学发病机制已得到公认，致病性自身抗体与靶抗原结合，引起一系列免疫炎症反应，如激活补体、炎细胞浸润、释放蛋白酶或溶酶体酶等，破坏在表皮细胞间或真表皮间黏附中起重要作用的结构分子，或直接影响结构分子的功能，导致表皮内或表皮下水疱、大疱的发生[2]。近年来该类疾病免疫学发病机理的研究取得了很大进展，明确寻常型天疱疮（PV）和落叶型天疱疮（PF）的主要靶抗原分别为桥粒芯糖蛋白3（Dsg3）和桥粒芯糖蛋白1（Dsg1），BP的主要靶抗原为大疱性类天疱疮抗原1（BPAG1，BP230）和大疱性类天疱疮抗原2（BPAG2，BP180）[3]，但是致病性自身抗体的来源及其产生机理尚不清楚。以往研究表明，T细胞依赖抗原（TD抗原）刺激B细胞产生抗体必需CD4+辅助性T细胞（Th）的辅助。抗体分泌细胞产生于外周淋巴器官（淋巴结、脾脏、扁桃体和派氏集合淋巴结等）的生发中心（Germinal Centers, GC），并在那里发生体细胞高频突变、抗体类别转换和高亲和力的选择。Th在GC的形成，选择高亲和力B细胞分化为记忆细胞或浆细胞，以及维持长时间的体液免疫应答等过程中都发挥着重要作用[4]。近期研究证实，体液免疫中辅助B细胞产生抗体的T细胞亚群为滤泡辅助性T细胞（T follicular helper cells,Tfh）。Tfh定位于淋巴滤泡，转录因子为Bcl-6，主要通过分泌白细胞介素21（IL-21）发挥作用，其膜表面高表达CXCR5、ICOS、PD-1、CD40L等，通过与B细胞表面相应的配体或受体相互结合辅助B细胞产生高亲和力抗体[5]。Tfh水平的异常与自身免疫性疾病的发病密切相关。在多种自身免疫性疾病如系统性红斑狼疮（SLE）、类风湿性关节炎（RA）、重症肌无力（MG）等患者或动物模型中均发现GC的形成和自身抗体的产生涉及Tfh细胞的异常。在小鼠模型中，阻断Tfh重要相关分子的功能，如ICOS、CD40L、IL-21等，则导致自身抗体产生下降[6-10]。鉴于目前自身免疫性疱病的自身抗体来源及产生机制尚不明确，而Tfh的主要功能是辅助B细胞产生高亲和力抗体，我们推测Tfh参与介导了自身免疫性疱病自身抗体的产生。本实验通过检测天疱疮和大疱性类天疱疮患者外周血中Tfh细胞水平，初步探索Tfh是否参与了ABD的发病，是否在ABD发病中辅助B细胞产生功能性抗体引起一系列免疫损伤，以期对ABD的发病机制进行进一步探索，并为ABD的治疗提供新的思路。目的：探索Tfh细胞与天疱疮和大疱性类天疱疮为代表的自身免疫性疱病的相关性。方法：收集天疱疮和大疱性类天疱疮患者及健康对照者外周血，提取外周血单个核细胞（PBMC）并分离血清，通过流式细胞术检测患者和正常对照者外周血中Tfh细胞水平，通过酶联免疫吸附试验（ELISA）检测患者和正常对照者血清中IL-21水平，分析Tfh和IL-21在患者与正常对照者中的差异以及与疾病严重程度的关系，分析Tfh与ABD发病的相关性。结果：大疱性类天疱疮患者外周血中Tfh水平高于健康对照者（中位数分别为11.25%和4.95%, P<0.001）；Tfh水平与反映病情严重程度的抗BP180-NC16A抗体滴度呈正相关（R=0.712, P<0.01）；大疱性类天疱疮患者血清中IL-21水平高于健康对照组（中位数分别为103.98pg/ml和46.77pg/ml,P<0.001），且IL-21水平与其血清中抗BP180-NC16A滴度呈正相关（R=0.578,P=0.030）；治疗后，随着患者病情的缓解及抗体滴度的下降，其外周血中Tfh水平较治疗前下降（中位数分别为10.50%和4.10%, P=0.003），治疗后血清中IL-21水平也较治疗前下降（中位数分别为99.98pg/ml和64.08pg/ml,P=0.012）。天疱疮患者外周血中Tfh水平高于健康对照组（中位数分别为10.00%和4.80%, P=0.001），血清中IL-21水平也高于正常对照组（中位数分别为72.84pg/ml和60.34pg/ml, P=0.011）；治疗后，随着患者病情的缓解，其外周血中Tfh细胞比例较治疗前下降（中位数分别为9.90%和5.00%，P=0.018），其治疗后IL-21水平较也治疗前下降（中位数分别为84.46pg/ml和69.58pg/ml,P=0.028）。结论：以天疱疮和大疱性类天疱疮为代表的ABD患者外周血Tfh细胞及其分泌的主要细胞因子IL-21水平均明显升高，，且BP患者中Tfh及IL-21水平均与反映病情严重程度的特异性抗体滴度呈正相关；治疗后，随着疾病缓解，患者外周血中Tfh及IL-21水平均显示出较治疗前下降趋势。以上结果提示Tfh可能在ABD发病过程中起着重要作用，且与疾病严重程度及活动度相关，通过本实验，为进一步探索ABD中自身抗体的来源提供了新的线索，进一步完善了ABD发病机制，并为ABD的治疗提供新的思路。

    Autoimmune bullous diseases (ABD) are a group of classic autoimmunediseases characterized by clinical erythema, blister, bulla and erosion. Commonlyseen in elderly individuals, they are famous of intractable and being tend torecurrent attacks. ABD is generally classified in two broad categories:intraepidermal and subepidermal bullous diseases. Pemphigus is most common inthe former group, and bullous pemphigoid is the typical representation of the latergroup[1].The immunological pathogenesis of ABD mediated by autoantibodies isgenerally accepted. It is confirmed that the binding of autoantibody to antigeninitiates a series of immune inflammatory events including activation ofcomplement, accumulation of inflammatory cell, release of proteinase andlysosomal enzyme that disrupts the critical adhesion molecules of epidermis orbasement membrane zone or affects the function of adhesion molecules directly,then leads to clinical blister or bulla[2].Recently advances were showed about thepathogenesis of ABD. It is clear that Dsg3and Dsg1are the major antigens ofpemphigus vulgaris and pemphigus foliaceus respectively and BPAG1(BP230)and BPAG2(BP180) are the antigen of bullous pemphigoid[3], but the source and emerging mechanism of autoantibody is not clear yet.Previous studies have shown that CD4+Th cells are required for B cellsprimed by T cell-denpendent antigen during the process of antibody production.Antibody secreting cells produced in germinal center (GC) where somatichypermutation, class switching of Ig and the choice of high affinity occurred. Thcells play a critical role in formation of GC, choice of high affinity B cellsdifferentiate into memory cells or plasma cells and maintain a prolonged humoralimmune response[4]. From recent studies it is believed that T follicular helpercells (Tfh) are responsible for helping B cells during antibody response. Tfhlocalize to the follicles, the transcription factor of them is Bcl-6, Tfh cellsexpressed high levels of CXCR5, ICOS, CD40L, OX40, PD-1, SAP, and so on,the combination of them with corresponding receptors or ligands on B cellssurface ensures Tfh help B cells produce high affinity antibodies. Tfh cellsproduce high levels of IL-21, by which they regulate the GC reactionappropriately[5].Recent studies support the involvement of Tfh cells in autoimmune diseases.Abnormal levels of Tfh cells were observed participating in GC formation andthe production of autoantibodies in many mice or patients with autoimmunediseases, including SLE, RA, MG and so on. Blocking the function of importantmolecules such as ICOS, CD40L and IL-21in Tfh resulted in reduced productionof antibodies[6-10]. Due to the source and mechanism of autoantibodies with ABDis not yet clear, and the major function of Tfh cells is help B cells to produceantibodies, by detecting the populations of Tfh cells in peripheral blood ofpatients with pemphigus and bullous pemphigoid, we explore preliminarywhether Tfh is involved in the incidence of ABD, seek further complement in thepathogenesis of ABD and find new ideas for the therapy of ABD. Objective: To explore the correlation of Tfh cells to ABD which representedby pemphigus and bullous pemphigoid.Methods: Peripheral blood was collected from pemphigus and BP patientsand normal controls. PBMC was extracted and serum was separated, doubleantibody sandwich ELISA was performed to measure the levels of serum IL-21,flow cytometry was performed to detect the level of Tfh in PBMCs, by which weexplore the correlation of Tfh cells to ABD.Results: The increase of Tfh cells in the peripheral blood of BP patients wasobserved compared with healthy controls (median11.25%versus4.95%,P<0.001). The populations of Tfh cells were positively correlated to the titer ofanti-BP180-NC16A in BP patients (R=0.712, P<0.01). Compared with healthycontrols, serum level of interleukin21(IL-21) was higher in the peripheral bloodof BP patients (103.98pg/ml versus46.77pg/ml, P<0.001). The levels of IL-21were positively correlated to the titer of anti-BP180-NC16A in BP patients(R=0.578, P=0.030). After therapy, accompanied by the remission of diseasesand decrease of titers of autoantibodies, the populations of Tfh cells weredescended compared with themselves before therapy (median10.50%versus4.10%, P=0.003). And the IL-21levels were declined too (median99.98pg/mlversus64.08pg/ml, P=0.012).Similarly, The increase of Tfh cells in the peripheral blood of pemphiguspatients was observed compared with healthy controls (median10.00%versus4.80%, P=0.001). Compared with healthy controls, serum level of IL-21washigher in the peripheral blood of pemphigus patients (median72.84pg/ml versus60.34pg/ml, P=0.011). After therapy, accompanied by the remission of diseasesand decrease of titers of autoantibodies, the populations of Tfh cells weredescended compared with themselves before therapy (median9.90%versus 5.00%，P=0.018); The IL-21levels were declined compared with themselvesbefore therapy too (median84.46pg/ml versus69.58pg/ml, P=0.028).Conclusion: Significant increase of Tfh cells populations were observed inpatients with pemphigus and BP, and IL-21, the major cytokine secteted by Tfhwas also increased compared with normal controls. Especially, Both of the Tfhpopulations and the IL-21levels show positive correlations with the titer ofanti-BP18NC16A autoantibodies in BP patients. After therapy, with the relief ofthe diseases, both of the levels of Tfh and IL-21were decreased compared withthemselves before therapy. These results suggest that Tfh cells play a critical rolein the autoantibody production of ABD and positively correlated with the diseaseseverity and activity. Our results provide new cues for searching of the source ofautoantibodies in ABD, add more mechanisms of ABD, and supply new ideasabout the therapy of ABD.

          滤泡辅助性T细胞（Tfh）与自身免疫性疱病的相关性研究

缩略语表5-7中文摘要7-10Abstract10-13前言14-16文献回顾16-41    一、自身免疫性疱病16-32    二、滤泡辅助性 T 细胞（Tfh）32-37    三、Tfh 细胞与自身免疫性疾病的关系37-411 材料41-432 方法43-463 结果46-574 讨论57-62小结62-63参考文献63-75个人简历和研究成果75-76致谢76

  本文关键词：滤泡辅助性T细胞（Tfh）与自身免疫性疱病的相关性研究，由笔耕文化传播整理发布。