胸腺中CXCR5+AChR+ B细胞亚群的功能及其与MG发病相关性的探讨

Myasthenia gravis (MG), a severe autoimmune disease, has no cure due to its unknown pathogenesis. The research in the pathogenesis of MG is one of hot topics in neurological and immunological area. Normal thymus has no germinal center, but in the thymus of 80% myasthenia gravis patients, ectopic germinal center was visible. Numerous studies suggest that the ectopic germinal centers in thymus are closely related to the pathogenesis of MG. In our previous studies, we discovered a subunit of B cells expressing both CXCR5 and AChR in the thymic ectopic germinal centers of MG patients with thymus hyperplasia for the first time. This B cell subunit didn't exist in thymuses of normal individuals. According to the antigen expression, antigen presentation and antibody production, we speculate that the CXCR5+AChR+ B cell subsets are related with the onset of MG. In the present study, we will comprehensively analyze their phenotype by flow cytometry, detect their antigen presentation and antibody production capacity by in vitro lymphocyte mixed culture tests, and explore their correlation with MG pathogenesis in the MG mouse model established by intraperitoneal injection of thymic single cell suspension. Our study aims to clarify the function of the B cell subsets and their functions in the pathogenesis of MG. This is of great significance to reveal the pathogenesis of MG.

重症肌无力是一种严重的自身免疫性疾病，发病机制不明，至今无法根治。对其发病机制的研究一直是神经病学及免疫学的研究热点。正常胸腺无生发中心，但80%的重症肌无力患者可出现胸腺异位生发中心，诸多研究提示胸腺异位生发中心与重症肌无力的发病密切相关。我们在前期研究中也发现胸腺异位生发中心具有免疫应答活性，并且首次发现重症肌无力合并胸腺增生患者胸腺异位生发中心存在CXCR5+AChR+ B细胞，而正常健康人胸腺组织中未发现该B细胞亚群，其在重症肌无力发病中的功能不明。本研究将采用流式细胞术对该B细胞亚群的表型进行较为全面的分析；采用体外淋巴细胞混合培养实验检测该B细胞亚群的抗原提呈和抗体产生能力；并使用重症肌无力患者胸腺组织单细胞悬液建立重症肌无力小鼠模型，探讨该B细胞亚群与重症肌无力发病的相关性。本研究以期阐明该B细胞亚群的功能及其在重症肌无力发病中的作用，对于揭示重症肌无力发病机制具有重要意义。

重症肌无力是一种严重的自身免疫性疾病，目前认为其发病机制与AchR抗体的产生有关。我们前期研究发现重症肌无力患者胸腺组织中存在CXCR5+AChR+ B细胞，而正常健康人胸腺组织中未发现该B细胞亚群，该B细胞亚群的功能尚不明确。本研究中，我们采用实时定量PCR、Western blot和免疫荧光法证实重症肌无力患者胸腺组织中存在CXCR5+AChR+ B细胞，而正常胸腺组织中未见该B细胞亚群。我们采用放射免疫分析法证实MG患者胸腺组织能产生AChR抗体，而正常胸腺组织未见AChR抗体。我们从胸腺切除术中获得新鲜胸腺组织，并依次采用交联有抗CXCR5的单克隆抗体、抗AChR的单克隆抗体mAb198（抗AChR单克隆抗体）和抗CD19单克隆抗体的免疫磁珠分选得到AChR+CXCR5+ B细胞，采用放射免疫分析法测定上清中AChR抗体的产生，结果显示经AChR刺激后，AChR+CXCR5+ B细胞能分泌AChR抗体，而未经AChR刺激时，几乎没有AChR抗体产生。结论：我们证实胸腺组织中存在能分泌AChR抗体的AChR+CXCR5+ B细胞，可能与重症肌无力的发病机制密切相关。