有丝分裂原体外活化成人外周血自然杀伤细胞CD69表达的研究

Study on the expression of CD69 on NK cells activated in vitro by mitogen from adult human peripheral blood

目的 :利用自然杀伤细胞 (NK)的早期活化标记CD69的表达探讨NK细胞体外活化的规律 ,促进这类细胞的临床应用 方法 :用Ficoll-Hypaque梯度离心法分离出正常成人外周血单个核细胞 (PBMC) ,分别在两种不同的有丝分裂原 ,植物血凝素 (PHA ,2 0 μg/mL)和佛波醇酯 (PDB ,10 - 7mol/L)存在的条件下进行培养 ,于 0、2、12、2 4h收获细胞后进行双色免疫荧光标记 ,以流式细胞术对NK细胞的CD69分子表达情况进行分析 结果 :NK细胞在PHA、PDB刺激 2h后CD69的表达明显上升 ,而且随时间的延长CD69的表达率逐渐增加 ,2 4h分别达到 ( 84 .96± 9.2 4 ) %、( 91 85± 2 .94 ) % ;PDB刺激组CD69的表达率在不同的时间点均比PHA组高 结论 :体外条件下 ,PHA、PDB均能快速上调NK细胞CD69表达 ,而且PDB( 10 - 7mol/L)作用明显强于PHA( 2 0 μg/mL)

Aim: To investigate the details of NK cells activation by mitogen in vitro with CD69 expression as activation marker for the application of these cells clinically. Methods: After separating the PBMCs from adult human peripheral blood by Ficoll-Hypaque centrifugation, the PBMCs culture procedure was applied to activate NK cells in vitro . These cells were activated by PHA(20 μg/mL)and PDB(10 -7 mol/L)separately. The expression rates of CD69 of 0, 2, 12, 24 h-cultured NK cells were estimated by flow cytometry following two-color immunofluorescent staining. Results: The expression rates of CD69 after 2 hours stimulation in response to PHA and PDB were (40.90±5.17)% and (62.82±7 69)%, and those after 24 hours were (84.96±9.24)% and (91.85±2.94)% respectively. The rates in response to PDB were higher than those of PHA at all time points. Conclusion: PHA、PDB may upregulate rapidly the expression of CD69 on NK cells in vitro and the effect of PDB(10 -7 mol/L)was stronger than that of PHA(20 μg/mL). [