miR-146a/Fas调控生发中心反应及参与类风湿关节炎发病的机制研究

A large amount of autoantibodies production depend on germinal center (GC) response in autoimmune pathogenesis. Altered GC response has been revealed in lymph nodes and synovium in rheumatoid arthritis(RA) patients, which is critical for sustained progress of this chronic disease.Our pilot studies have found that high levels of miR-146a in CD4+ T cells and B cells purified from synovial fluid of RA. Moreover,we found incresed sizes of GCs and more GC B cells in spleen and lymph nodes in miR-146a transgenic mice. We also identified that Fas is the specific target gene of miR-146a in GC B cells. Based on these findings, we hypothesize that miR-146a/Fas maybe regulate GC response and finally involve in the secretion of autoantibody in RA.In this study,we will determine the molecular mechanisms underlying miR-146a-mediated GC formation,GC B cell differentiation and function. Next, Using a mouse K/BxN model with autoimmune arthritis, we will investigate whether and how GC B cells with miR-146a-overexpressing contribute to GC response and autoimune progression. Furthermore, we will examine the role of miR-146a/Fas in modulating GC B cell functions from RA patients and normal subjects and elucidate the molecular mechanisms involve in synovial inflammation in RA. Together, our proposed studies will provide further insight in understanding autoimmune progression and may facilitate the development of new treatment strategy for RA.

类风湿关节炎（RA）患者淋巴结和滑膜异常的生发中心（GC）反应，分泌大量高亲和力的自身抗体，是导致炎症持续存在和慢性化的重要因素。课题组前期研究发现RA滑液CD4+ T细胞和B细胞均高表达miR-146a，miR-146a转基因小鼠呈现生发中心增多、GCB细胞明显增加的特征，并鉴定Fas是miR-146a在GCB细胞中特异的靶基因，提示miR-146a/Fas可能调控了生发中心反应，并最终参与了RA中自身抗体的形成。为证实这一假说，我们拟开展如下研究：以转基因小鼠为模型，分析miR-146a/Fas调节GC反应的特点及机制；以K/BxN小鼠和RA病人为研究对象，探讨miR-146a/Fas对GC反应和GCB功能的影响，并分析调节的分子网络，进一步阐明miR146a/Fas调控GC反应并参与RA发病的分子机制。本研究的完成有助于深入理解RA的免疫病理机制，并为RA的治疗策略提供了新的线索。

B细胞参与了类风湿关节炎发病和炎症病理机制的各个环节，miRNAs是调节B细胞增殖和功能的重要分子。我们的前期研究发现miR-146a调节类风湿关节炎中B细胞的功能，在miR-146a 转基因小鼠中，生发中心B细胞显著增加，但其具体作用机制尚不清楚。本研究中，我们进一步探讨了miR-146a调节RA病人生发中心反应的机制，我们的研究发现，在转基因小鼠中，IgE的分泌显著增加，而IgM和IgG的抗体亲和力成熟无明显改变。进一步通过TMT法定量分析了转基因小鼠和野生小鼠B细胞蛋白组表达差异，研究发现miR-146a上调了Ig类别转换相关蛋白14-3-3σ的表达水平；而是miR-146a的新靶标Smad4的表达下降；进一步在OVA免疫的小鼠模型的研究中，我们发现小鼠体内OVA特异性的IgE抗体增加。这些结果证实miR-146a通过调节14-3-3σ的表达水平，促进了IgE的类别转换和分泌。上述研究提示，miR-146a直接参与了生发中心反应和B细胞分泌抗体的功能，为自身免疫疾病治疗的新策略提供了线索。