RasGRP3调控Ras信号通路异常活化系统性红斑狼疮B细胞及其机制的研究

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies involving multiple organs and tissues, the endogenous hypersensitive B lymphocyte is an important part in lupus pathogenesis. Preliminary results of our project confirmed that RasGRP3 expression in peripheral blood mononuclear cells and B cells of SLE patients was higher than that of normal; The stimulation of B cells from SLE patients with glucocorticoids in vitro could decrease RasGRP3 expression, suggesting that RasGRP3 might play an important role in autoimmune B cell activation of SLE. Accordingly, we plan to use RNA interference to knock down RasGRP3 expression of B cells from SLE patients, build up RasGRP3 vector transfected to B cells of normal human to increase its higher expression for exploring its role in B cell activation, as well as using pulldown ，gold nanoclusters tracing and other methods to investigate RasGRP3 phosphorylation , its location and the activation of Ras family members of its downstream in B cells, for exploring the effect and mechanism of RasGRP3 by Ras/ERK signaling pathway involved in B cells activation in SLE by using lupus mice model, in order to put forward a new interpretation of autoimmune mechanism in SLE and provide new ideas of its immunological therapy.

系统性红斑狼疮（SLE）是以自身抗体的产生为特征的累及多个器官和组织的自身免疫性疾病，内源性的高反应性B淋巴细胞是狼疮发病的重要环节。本课题组前期研究证实RasGRP3在SLE患者外周血单个核细胞以及B细胞中表达均较正常人高；体外用糖皮质激素刺激SLE患者B细胞可下调RasGRP3的表达，推测RasGRP3可能在SLE的自身免疫B细胞活化中扮演重要作用。据此，本课题设计采用RNA干扰技术降低SLE患者B细胞RasGRP3的表达，并构建RasGRP3载体转染至正常人B细胞使之高表达，研究其对B细胞活化的作用，以及采用pulldown及金纳米簇示踪等方法，研究RasGRP3在B细胞中磷酸化、定位和下游Ras家族成员的活化的作用，进一步用狼疮鼠模型探讨SLE中RasGRP3经Ras/ERK信号通路参与活化B细胞的作用及其机制，为SLE自身免疫发病机制提出新的解读，对其免疫学干预治疗提供新的思路。

系统性红斑狼疮（SLE）是以自身抗体的产生为特征的累及多个器官和组织的自身免疫性疾病，内源性的高反应性B淋巴细胞是狼疮发病的重要环节。我们证明，SLE患者及狼疮模型鼠B细胞中RasGRP3的表达均高于正常人或正常鼠。而体内外实验均表明糖皮质激素治疗后B细胞中RasGRP3的表达明显降低。RasGRP3的表达与Erk和Akt的活化紧密相关。当用siRNA转染方法沉默狼疮鼠B细胞RasGRP3表达后，Raf/Mek/Erk通路和PI3K/Akt通路的活性明显降低，IL-6和TNFα等炎性细胞因子分泌减少，且细胞凋亡增加。同时，我们也发现RasGRP3在B细胞中磷酸化位点为thr133。 说明RasGRP3是系统性红斑狼疮自身免疫B细胞活化的重要的促进因子。该研究丰富了糖皮质激素治疗狼疮的作用机制并为将来SLE的生物学治疗提供新的思路与靶点。为采用金纳米簇对RasGRP3进行示踪，我们国内外首次对牛血清白蛋白包封的金纳米簇进行了系统、全面的体内外毒性研究，为疾病的诊断与靶向治疗提供重要的载体与依据。