摘要
目的：建立基于流式细胞术的评价单核细胞介导的抗体依赖性细胞介导的细胞毒效应的检测方法。方法 PKH26和CFSE染色的P815细胞为靶细胞，与P815特异性抗体孵育形成抗原抗体复合物，加入外周血单个核细胞作为效应细胞，共同孵育后流式细胞术检测 CD3－CD14＋PKH26＋CFSE－细胞群的百分比，并确定最佳效靶比及效应细胞和靶细胞的孵育时间。运用上述方法对23例HCV慢性感染者和22例健康人的单核细胞介导的抗体依赖性细胞毒作用（ antibody depend-ent cellular cytotoxicity ， ADCC）进行比较分析。结果可通过流式细胞技术检测CD3－CD14＋PKH26＋CFSE－细胞群来评价单核细胞介导的ADCC效应，最佳效靶比为10∶1，最佳杀伤孵育时间为4 h。慢性HCV感染者单核细胞介导的ADCC效应较健康对照明显降低（ P＝0．009）。结论本研究建立了基于流式细胞术的单核细胞介导的ADCC效应的检测方法，为病毒感染及药物研发中免疫学评价提供快速、敏感、安全的检测手段。
Objective To establish a flow cytometry-based assay for the detection of monocyte-me-diated antibody-dependent cell-mediated cytotoxicity （ ADCC ） .Methods P815 cells double stained with PKH26 and carboxyfluorescein succinimidyl ester （ CFSE ） were used as target cells and coated with P 815 specific antibodies to form antigen-antibody complexes .The peripheral blood mononuclear cells were isolated as effector cells and co-cultured with the antigen-antibody complexes .The CD3-CD14＋PKH26＋CFSE-cell population were gated by flow cytometry .Optimized effector/target cell ratio and incubation time for killing assay were identified .Monocyte-mediated ADCC in 23 patients with chronic HCV infection and 22 healthy subjects were analyzed .Results The monocyte-mediated ADCC could be evaluated through analyzing the CD3-CD14＋PKH26＋CFSE-cells with flow cytometry .The optimized effector/target cell ratio was 10 ∶1 and the optimized time for incubation was 4 h.Monocyte-mediated ADCC was inhibited in patients with chronic HCV infection as compared with healthy subjects （P=0.009）.Conclusion A flow cytometry-based assay for the detection of monocyte-mediated ADCC was established , which could be used as a fast , sensitive and safety method for the evaluation of monocyte-mediated ADCC during viral infections and the research and de-velopment of drugs .
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第1期18-22,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金项目（31100126,81271826）
国家科技重大专项（2012ZX10001008,2014ZX10001001-002,2012ZX10004501-001-006）
北京市自然科学基金面上项目（7122108）
传染病预防控制国家重点实验室项目（2012SKLID103,2011SKLID207）
