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目的:观察系统性红斑狼疮(SLE)患者B细胞表面功能分子表达的特征及其功能状态,评价以FcγRⅡB1(CD32)为代表的B细胞自身抑制调节机制在SLE发病中的作用。方法:采用Ficoll密度梯度离心法分离出人外周血单个核细胞(PBMC),并以免疫磁珠法(MACS)分离纯化B细胞。采用荧光分光光度法检测B细胞受不同激活物刺激后细胞内钙([Ca2+]i)的反应。用ELISA法检测B细胞与刺激物共同培养后所分泌IgG的量。采用流式细胞术及间接免疫荧光染色法,检测B细胞膜表面CD32、CD19及IgM的表达水平。结果:(1)以羊抗人μ链的F(ab′)2片段及完整IgG分别刺激SLE患者B细胞时,其[Ca2+]i反应的比值显著低于类风湿性关节炎(RA)患者(P<0.05)及正常人对照(P<0.01)。(2)分别用葡萄球菌A蛋白(SPA)单独刺激与SPA和羊抗人μ链的完整IgG抗体共同刺激SLE患者的B细胞所分泌的IgG的比值,明显低于RA患者及正常人对照组(P<0.05)。(3)SLE患者与RA患者及正常对照组B细胞上CD19、CD32及IgM的表达无统计学意义(P>0.05)。结论:SLE患者B细胞上CD32抑制性信号传导的异常,可能是导致B细胞过度活化的重要机制。
OBJECTIVE: To observe the features and functional status of B cell surface functional molecules expression in patients with systemic lupus erythematosus (SLE) and to evaluate the role of B cell autoregulation regulation mechanism represented by FcγRⅡB1 (CD32) in the pathogenesis of SLE. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation and purified by immunomagnetic beads method (MACS). Fluorescence spectrophotometry was used to detect the intracellular calcium ([Ca2 +] i) response of B cells stimulated by different activators. The amount of IgG secreted after co-culture of B cells and stimulators was measured by ELISA. Flow cytometry and indirect immunofluorescence staining were used to detect the expression of CD32, CD19 and IgM on the surface of B cell membrane. Results: (1) The ratio of [Ca2 +] i response was significantly lower in patients with SLE when stimulated with F (ab ’) 2 fragment of goat anti-human μ chain and intact IgG than those in patients with rheumatoid arthritis (P <0.05) and normal controls (P <0.01). (2) The ratio of IgG secreted by B cells stimulated by Staphylococcus A protein (SPA) alone and SPA and goat anti-human chain together to stimulate SLE patients was significantly lower than that of RA patients and normal controls (P <0.05). (3) The expression of CD19, CD32 and IgM on B cells in SLE patients and RA patients and normal control group was not statistically significant (P> 0.05). Conclusion: Abnormality of CD32 inhibitory signal transduction on B cells in SLE patients may be an important mechanism leading to over-activation of B cells.