补体C5a受体拮抗剂体外筛选平台的建立

摘要

Objective To estahlish the stable methodology f'or screening antagonists of CD88, one of C5a receptors. Methods Whole blood from volunteers was collected and then stimulated by C5a with different concentration for 10 -30 min in the presence or absence of PMX-53 and LPS. Flow cytometry was used to detect the expression of CD11b which on the surface of neutrophil. Other functions of nentrophil , including lysozyme release ,oxidative burst and interleukin (IL)-8 production , were also examined respectively. Finally , western blotting was used to detect the content of total and phosphorylated ERK and AKT. Thus, the effects about C5a receptor stimulant and the positive medicine PMX-53 on biochemical indicators were investigated. Results The C5a stimulation actively up-regulated CD11b expression,evoked lysozyme and reactive oxygen species release,increased the IL-8 production and the contents of total and phosphorylated ERK/AKT. While the positive medicine PMX-53 significantly blocked these effects. Conclusion The methodology in vitro f'or screening C5a receptor antagonists in human whole blood is successfully established.%目的建立稳定的补体C5a受体(C5aR)拮抗剂筛选平台.方法采集人外周静脉抗凝血,与不同浓度C5a、脂多糖及PMX-53孵育10～30min.通过流式细胞术检测中性粒细胞表面CD11b的表达,溶菌酶检测试剂盒观察中性粒细胞分泌溶菌酶的能力变化,罗丹明-123检测中性粒细胞呼吸爆发的变化,ELISA检测细胞上清液中白细胞介素(IL)-8的表达变化,Western Blotting检测胞外信号调节激酶(ERK)、蛋白激酶B(AKB或AKT)含量及其磷酸化水平的变化,考察补体C5a刺激及使用阳性药物PMX-53后对各生化指标的影响.结果 C5a刺激人外周血可增强中性粒细胞CD11b的表达,促进溶菌酶释放和IL-8的分泌,激发呼吸爆发,上调ERK、AKT的含量和磷酸化水平,阳性药物PMX-53则能显著抑制C5a的上述生物学效应.结论成功建立C5aR拮抗剂人全血体外筛选平台,为C5aR拮抗剂筛选及功能研究奠定了基础.