MG患者外周血Th17细胞与AChR抗体产生的关系研究  

The circulating Th17 cells enhance AChR antibody production in myasthenia gravis patients

作　　者：张晓燕[1] 常婷[2] 杨林[3] 刘沙沙[2] 李柱一[2] YANG Kun ZHANG Xiaoyan CHANG Ting YANG Lin LIU Shasha YANG Kun LI Zhuyi(Department of Neurology, Tangdu Hospital, the Fourth Military Medical University, Xi ＇an Shaanxi Province 710038, China)

机构地区：[1]中国人民解放军兰州总医院神经内科,730050 [2]第四军医大学唐都医院神经内科,710038 [3]中国人民解放军兰州军区善后办机关门诊部,730000

出　　处：《中国神经免疫学和神经病学杂志》2017年第2期105-109,共5页Chinese Journal of Neuroimmunology and Neurology

基　　金：国家自然科学基金资助项目(31270952);甘肃省自然科学基金资助项目(1506RJZA302)

摘　　要：目的探讨重症肌无力(MG)患者外周血中Th17细胞及相关细胞因子白细胞介素17(IL-17)在MG发病中的作用。方法收集40例MG患者和10名健康人(对照组)外周血标本,采用流式细胞术检测外周血单个核细胞(PBMCs)中Th17细胞比例,反转录酶-聚合酶链锁反应(RT-PCR)检测PBMCs中维甲酸受体相关孤儿受体γt(RORγt)mRNA水平,ELISA检测血清中IL-17水平,放射免疫沉淀法检测血清中抗乙酰胆碱受体抗体(AChR-Ab)滴度;分离PBMCs中CD4^+T细胞和CD19^+B细胞与金黄色葡萄球菌肠毒素B(SEB)进行共培养,培养系统中加入人IL-17和(或)IL-21中和抗体,放射免疫测定法检测培养液中AChR-Ab滴度。采用MG评分(quantitative MG scoring system,QMGs)对MG的严重程度进行评估,并对MG患者的Th17细胞比例、RORγt mRNA和IL-17水平与病情QMGs的相关性,以及MG患者抗AChR-Ab滴度与PBMCs中Th17细胞比例的相关性进行分析。结果 MG患者PBMCs中Th17细胞比例[1.11%(0.90%,1.34%)]高于健康对照组Th17细胞比例[0.26%(0.08%,0.36%)](z=5.494,P<0.001),且与疾病严重程度呈正相关(r=0.4394,P=0.0046);血清中IL-17水平和PBMCs中RORγt mRNA相对表达[分别71.46(53.91,104.76)pg/mL、2.63(1.94,3.12)]均较健康对照组[分别18.82(12.73,29.80)pg/mL、1.13(0.98,1.28)]显著增高(均P<0.001);MG患者血清中抗AChR-Ab滴度[2.34(1.19,3.60)nmol/L]较健康对照组[-0.08(-0.24,-0.03)nmol/L]显著增高(z=4.662,P<0.001),且与Th17细胞比例呈正相关(r=0.7066,P=0.0001)。MG患者外周血T、B细胞与SEB共培养后抗AChR-Ab水平高于未加入SEB时及健康对照(均P<0.01);加入抗人IL-21或IL-17中和抗体后,两者AChR-Ab滴度与未加入抗体时AChR-Ab滴度比较均降低(均P<0.05),且均仍高于MG患者未加入SEB时及健康对照(P<0.01);在培养上清中同时加入抗人IL-21和IL-17中和抗体时AChR-Ab滴度明显低于加入单种抗体时,而与未加入SEB时及健康对照差异无统计学意义(均P>0.05)。结论 MG患者外周血中Th17细Objective To investigate the role of circulating Th17 cells and IL-17 in myasthenia gravis（MG）.Methods Forty MG patients and 10 healthy controls（HC）were recruited.The proportion of Th17 cells in peripheral blood mononuclear cells（PBMCs）was detected via flow cytometry.The transcription factor RORγt mRNA expression in PBMCs was detected with Reverse Transcription-Polymerase Chain Reaction（RTPCR）.The concentration of cytokine IL-17 in sera was measured by ELISA.The anti-human AChR antibody in the serum was assayed using the radioimmunoprecipitation method.CD19~＋B cells and CD4~＋T cells were isolated from PBMCs of MG patients and healthy controls at the presence of SEB.The anti-AChR antibody secretion in T/B cells co-culture supernatant was detected by radioimmunoprecipitation.The effects of inhibiting IL-17 or IL-21 were examined by adding anti-IL-17 or IL-21 neutralizing antibody in the co-culture system.A quantitative MG scoring system（QMG score）was used to objectively evaluate the disease severity.Then,we analyzed the correlation between Th17 cells proportion,serum IL-17 level,RORγt mRNA and QMG scores.Results The percentage of Th17 cells increased significantly in PBMCs of MG patients compared with HC（P〈0.0001）,and Th17 cells counts was positively correlated with disease severity（r=0.4394,P =0.0046）.Th17 cellassociated transcription factors RORγt mRNA and cytokines IL-17 expression elevated in MG patients（P〈0.001）.The titers of anti-AChR antibody（AChR-Ab）increased significantly in MG patients compared with HC（P〈0.001）,and the titer of AChR-Ab was positively correlated with disease severity.High level of antiAChR antibody was detected in the co-culture of patients T and B cells.Blocking IL-17 or IL-21 alone by neutralizing antibodies led to a moderate decrease of anti-AChR antibody titer,while simultaneous blockage of IL-17 and IL-21 resulted in dramatically decrease of anti-AChR antibody titer.Conclusions In creased Th17 cell was involved in the pathological p

关 键 词：重症肌无力 Th17淋巴细胞 IL-17 胆碱能 抗体 

分 类 号：R746.1[医药卫生—神经病学与精神病学]