分泌IFN-γ的杀伤性树突状细胞的体外诱导培养及鉴定  

Inducing Culture in Vitro and Identification of IFN- γ Producing Killer Dendritic Cells

作　　者：钱莉[1] 陆家辉[1] 潘兴元[1] 田芳[1] 龚卫娟[1] 季明春[1] 

机构地区：[1]扬州大学医学院病原生物学与免疫学教研室,江苏扬州225001

出　　处：《昆明医学院学报》2011年第6期4-7,共4页Journal of Kunming Medical College

基　　金：国家自然科学基金资助项目(81001308);江苏省自然科学基金资助项目(BK2010315)

摘　　要：目的从体外诱导的骨髓来源的树突状细胞(dendritic cells,DC)中分离分泌IFN-γ的杀伤性树突状细胞(IFN-γproducing killer dendritic cells,IKDC).方法取C57BL/6小鼠骨髓细胞,以小鼠重组GM-CSF和IL-4协同诱导下培养.培养第6天,加LPS刺激.培养第7天,收集细胞,通过流式细胞仪(flowcytometer,FCM)分选出CD11clowB220+NK1.1+的细胞,FCM进一步鉴定其表型.并将其与肿瘤细胞系B16F10共培养24 h后,CBA(cytometric bead array)法检测共培养上清中IFN-γ的分泌水平.结果体外培养的骨髓来源的DC中,FCM检测存在CD11clowB220+NK1.1+的细胞,进一步鉴定发现其高表达CD49b,低表达MHC II类分子,不表达Gr-1分子;并且与肿瘤细胞B16F10共培养后可分泌大量IFN-γ.结论通过FCM分选的方法可从体外培养的骨髓来源的DC中获得IKDC.Objective To isolate the IFN-γ producing killer dendritic cells（IKDC）from bone marrow-derived dendritic cells.Methods Bone marrow cells were extracted from the tibia and femur bones of C57BL/6 mice and cultured in the RPMI 1640 medium with recombinant mouse granulocyte-macrophage colony-stimulating factor（rmGM-CSF）and interleukin（IL）-4 in vitro.At day 6,lipopolysaccharide（LPS）was added to promote DCs＇ maturation and differentiation.At day 7,CD11clowB220＋NK1.1＋ cells were generated from bulk DC by FCM sorting.Expression of CD49b,MHC class II and Gr-1 was assessed using appropriate conjugated mAb.Furthermore,CD11clowB220＋NK1.1＋ cells were stimulated with B16F10 tumor cells and IFN-γ levels were measured in the supematants using CBA kits.Results CD11clowB220＋NK1.1＋cells were found in cultured bone marrow-derived dendritic cells,and these cells with IKDC phenotype highly expressed CD49b but lowly expressed MHC class II,and did not express Gr-1.Furthermore,FCM-sorted CD11clowB220＋NK1.1＋ cells could produce IFN-γ in response to B16F10 tumor cells.Conclusion IKDC can be isolated from bone marrow-derived DC by FCM sorting.

关 键 词：杀伤性树突状细胞 骨髓 细胞培养 

分 类 号：R392.2[医药卫生—免疫学]