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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 a stable C5a receptor antagonist screening platform. Methods Peripheral anticoagulants were collected and incubated with different concentrations of C5a, lipopolysaccharide and PMX-53 for 10-30 min. The expression of CD11b on neutrophils was detected by flow cytometry. The lysozyme detection kit was used to observe the changes of neutrophil secreting lysozyme. Rhodamine-123 was used to detect the change of neutrophil respiratory burst. The expression of interleukin-8 (IL-8) was detected by Western Blotting. The levels of ERK and AKB or AKT were detected by Western Blotting. The Influence of PMX-53 on Biochemical Indexes. Results C5a stimulation of human peripheral blood can enhance neutrophil CD11b expression, promote the release of lysozyme and IL-8 secretion, stimulate respiratory burst, upregulate ERK, AKT levels and phosphorylation, the positive drug PMX-53 can significantly Inhibit the above biological effects of C5a. Conclusion The human whole blood screening platform of C5aR antagonist has been successfully established, which lays the foundation for the screening of C5aR antagonist and its function.