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目的:检测脾脏和淋巴结中B细胞活化相关蛋白表达及蛋白磷酸化表达水平的变化,从B细胞活化的角度探讨Tα146-162-iMDC干预实验性自身免疫性重症肌无力(EAMG的作用机制。方法:采用树突状细胞(DC)负载Tα146-162干预实验性自身免疫性重症肌无力小鼠(EAMG)的发病,34只6～8周龄健康雄性C57BL/6J小鼠,随机分为模型组(A)、干预组(B)和对照组(C)。体外培养DC,然后负载Tα146-162进行干预。从初次免疫起至第90天实验终止前,行EAMG严重性临床评估及发病率的计算。Western blot检测Syk、Lyn、Btk和PLC-γ2蛋白及蛋白磷酸化表达。结果:临床评估:A组发病率高于B组(75%vs25%,P<0.05)。实验终止时2组临床评分分别为1.69±1.12vs0.35±0.67(P<0.01)。C组小鼠无发病。A组小鼠的脾脏和淋巴结Syk和PLC-γ2蛋白表达和磷酸化水平较C组小鼠升高(P<0.01),B组较A组下降(P<0.05),但高于C组(P<0.05);A组小鼠的脾脏和淋巴结Lyn蛋白表达和磷酸化水平较C组小鼠降低(P<0.01),B组较A组升高(P<0.05),但仍低于C组(P<0.05);A组小鼠的脾脏和淋巴结Btk蛋白表达较C组小鼠升高(P<0.01),B组较A组降低(P<0.05),仍高于C组(P<0.05),但磷酸化水平三组无统计学意义(P>0.05)。结论:Tα146-162-iMDC干预,能显著降低EAMG的发病率,改善临床症状,其机制可能与抑制B细胞活化有关。
Objective: To investigate the changes of B cell activation-related protein expression and protein phosphorylation in spleen and lymph nodes, and to explore the mechanism of Tα146-162-iMDC intervention on experimental autoimmune myasthenia gravis (EAMG) from the perspective of B cell activation. : To investigate the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) using Tα146-162 loaded with dendritic cells (DCs). Thirty-four healthy male C57BL / 6J mice aged 6-8 weeks were randomly divided into model group (A), intervention group (B) and control group (C). DCs were cultured in vitro and then interfered with Tα146-162.The clinical evaluation and incidence of EAMG severity before the termination of the experiment from the initial immunization to the 90th day Western blot was used to detect the protein and protein phosphorylation of Syk, Lyn, Btk and PLC-γ2.Results: The clinical evaluation showed that the incidence of group A was higher than that of group B (75% vs 25%, P <0.05) The clinical scores were 1.69 ± 1.12vs0.35 ± 0.67 (P <0.01) .The mice in group C had no disease.The expression and phosphorylation of Syk and PLC-γ2 in spleen and lymph node of group A were higher than those in group C (P <0.01). The B group was lower than the A group (P <0.05), but higher than the C group (P <0.05). The spleen and lymph node Ly (P <0.01). The B group was higher than that of A group (P <0.05), but still lower than that of C group (P <0.05). The spleen (P <0.01), B group was lower than A group (P <0.05), and still higher than C group (P <0.05), but the phosphorylation level of the three groups had no statistical significance (P> 0.05) .Conclusion: Tα146-162-iMDC intervention can significantly reduce the incidence of EAMG and improve clinical symptoms, and its mechanism may be related to the inhibition of B cell activation.