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为阐明可溶性的抗CD47单克隆抗体(B6H12)对树突状细胞(DC)的免疫调控作用及分子机理.联合应用 rhGM-CSF、IL-4、细菌脂多糖(LPS)在体外诱导扩增人外周血单核细胞来源的DC,并在培养体系中添加抗CD47单 克隆抗体(B6H12);采用透射电镜观察DC形态,流式细胞术检测DC膜表面分子的表达;ELISA法检测DC释放的 IL-12 P70水平;BrdU-ELISA法检测DC刺激同种异型淋巴细胞增殖,凝胶电泳迁移率改变实验(EMSA)检测NF-κB 结合活性变化。结果发现:①经抗CD47单克隆抗体(B6H12)处理的DC,膜表面标记(CD80、CD86、CD1a、CD83、 DR)的表达显著地低于未加B6H12单克隆抗体DC组(P<0.05);其释放IL-12 P70蛋白质的能力及刺激同种异 型淋巴细胞增殖的能力也显著低于对照组(P<0.01)。②与未加B6H12单克隆抗体DC组相比,B6 H12单克隆抗 体处理DC组的NF-κB活性显著降低(P<0.05),并且这种抑制作用随单克隆抗体浓度增大而增强。结论:可溶 性的抗CD47单克隆抗体通过抑制NF-κB的结合活性,影响着DC向成熟发育及功能。
To clarify the immunomodulatory effect and the molecular mechanism of soluble anti-CD47 monoclonal antibody (B6H12) on dendritic cells (DCs), the recombinant human rhGM-CSF, IL-4 and bacterial lipopolysaccharide (LPS) DCs derived from peripheral blood mononuclear cells were cultured in vitro. Anti-CD47 monoclonal antibody (B6H12) was added into the culture system. The morphology of DCs was observed by transmission electron microscopy. The expression of DCs on DCs was detected by flow cytometry. -12 P70. The proliferation of allogeneic lymphocytes stimulated by DC was detected by BrdU-ELISA. The change of NF-κB binding activity was detected by electrophoretic mobility shift assay (EMSA). The results showed that: (1) The expression of CD80, CD86, CD1a, CD83, DR on DCs treated with anti-CD47 monoclonal antibody (B6H12) was significantly lower than that in DCs without B6H12 monoclonal antibody (P < The ability of releasing IL-12 P70 protein and the ability of stimulating allogeneic lymphocyte proliferation were also significantly lower than those of the control group (P <0.01). ② The NF-κB activity of DCs treated with B6-H12 monoclonal antibody was significantly lower than that of DCs without B6H12 monoclonal antibody (P <0.05), and the inhibitory effect was enhanced with the increase of monoclonal antibody. Conclusion: The soluble anti-CD47 monoclonal antibody can affect the development of DC to mature and its function by inhibiting the binding activity of NF-κB.