分泌IFN-γ的杀伤性树突状细胞的体外诱导培养及鉴定

摘要

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+NK 1.1 + 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.%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-Υproducing 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,KCM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 bwad 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.