补体C5a受体拮抗剂体外筛选平台的建立

Establishment of screening platform in vitro for C5a receptor antagonists

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10～30 min。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。

Objective To establish the stable methodology for 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 for screening C5a receptor antagonists in human whole blood is successfully established.