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目的体外模拟γδT细胞受流感病毒刺激后的活化情况及分化方向,探讨活化后的γδT细胞对经流感病毒感染的人外周血来源的巨噬细胞(MDM)的杀伤作用。方法分离6例正常志愿者人外周血单个核细胞(PBMC),经H1N1、H3N2、BV分别体外刺激12～24h,流式细胞技术检测刺激前后γδT细胞活化标志CD25和CD69,以及细胞分化标志CD4、CD8;采用LDH杀伤实验研究流感病毒刺激后γδT细胞对感染的MDM细胞的杀伤作用。结果与刺激前组相比(CD25:19.9%,CD69:11.1%),受流感病毒刺激各组γδT细胞均出现CD25和CD69活化,以CD69活化为主(CD25 H1N1:58.2%,H3N2:50.8%,B型流感:49.5%;CD69H1N1:61.4%,H3N2:47.0%,B型流感:30.5%),且H1N1刺激后活化指标显著高于其他两组;刺激前γδT细胞主要表现为CD4-CD8-,受各亚型流感病毒刺激后,γδT细胞趋向于发展为CD4+CD8+亚型(刺激前:14.4%,H1N1:33.8%,H3N2:29.7%,B型流感:29.1%);LDH杀伤实验证实刺激后γδT细胞均体现对流感感染MDM细胞的杀伤作用,甲型流感组H1N1(35.46%～52.17%)和H3N2(28.46%～41.34%)刺激后的杀伤作用显著高于B型流感(18.94%～25.98%);甲型流感刺激组(H1N1和H3N2组)存在一定程度的交叉杀伤作用。结论γδT细胞能被各型流感病毒快速活化分化成功能性的CD4+CD8+细胞,且发挥杀伤细胞的杀伤和交叉杀伤功能。
Objective To investigate the activation and differentiation of γδT cells stimulated by influenza virus in vitro and to investigate the killing effect of activated γδT cells on human peripheral blood mononuclear cells (MDM) infected with influenza virus. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 6 normal volunteers. The cells were stimulated by H1N1, H3N2 and BV for 12-24 h respectively. The activation markers CD25 and CD69 of γδ T cells before and after stimulation were detected by flow cytometry. , CD8. The cytotoxicity of γδT cells on MDM cells infected by influenza virus was studied by LDH killing assay. Results CD25 and CD69 were activated in all the groups of γδT cells stimulated by influenza virus (CD25: H1N1: 58.2%, H3N2: 50.8% , Influenza B (49.5%), CD69H1N1 (61.4%), H3N2 (47.0%) and influenza B (30.5%). The activation index of H1N1 was significantly higher than that of the other two groups. , ΓδT cells tended to develop CD4 + CD8 + subtypes (14.4% before stimulation, H1N1: 33.8%, H3N2: 29.7%, and B type influenza: 29.1%) after being stimulated by each subtype of influenza virus; The cytotoxicity of γδT cells to MDM cells infected by influenza A was significantly higher than that of influenza B (18.94%) after being stimulated by H1N1 (35.46% -52.17%) and H3N2 (28.46% -41.34% ~ 25.98%). Influenza A (H1N1 and H3N2) had some cross-killing effects. Conclusion γδT cells can be rapidly activated and differentiated into functional CD4 + CD8 + cells by various influenza viruses, and kill and cross-kill killer cells.