CAJ | 학술논문

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。
목적：건립기우류식세포술적평개단핵세포개도적항체의뢰성세포개도적세포독효응적검측방법。방법 PKH26화CFSE염색적P815세포위파세포，여P815특이성항체부육형성항원항체복합물，가입외주혈단개핵세포작위효응세포，공동부육후류식세포술검측 CD3－CD14＋PKH26＋CFSE－세포군적백분비，병학정최가효파비급효응세포화파세포적부육시간。운용상술방법대23례HCV만성감염자화22례건강인적단핵세포개도적항체의뢰성세포독작용（ antibody depend-ent cellular cytotoxicity ， ADCC）진행비교분석。결과가통과류식세포기술검측CD3－CD14＋PKH26＋CFSE－세포군래평개단핵세포개도적ADCC효응，최가효파비위10∶1，최가살상부육시간위4 h。만성HCV감염자단핵세포개도적ADCC효응교건강대조명현강저（ P＝0．009）。결론본연구건립료기우류식세포술적단핵세포개도적ADCC효응적검측방법，위병독감염급약물연발중면역학평개제공쾌속、민감、안전적검측수단。
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 .