小鼠骨髓来源树突状细胞的分离与扩增培养

目的 :探讨树突状细胞 (DC)分离纯化及其体外扩增的方法。方法 :无菌制备C57BL/6小鼠骨髓 ;依次用红细胞裂解液去除红细胞 ,通过半粘附法去除T、B细胞 ,又在粒细胞巨噬细胞集落刺激因子 (GM CSF)和白细胞介素 4 (IL 4 )协同诱导下培育 ,DC前体分化发育成DC并扩增。在第 7天用脂多糖 (LPS)和肿瘤坏死因子 a (TNF a)刺激 4 8h ,检测细胞因子白介素 12 (IL 12 )浓度及细胞表面标志CD11c、CD80、CD86和MHCⅡ。结果 :DC细胞数增加 ,其形态在光镜下多为特征性星形 ,也有梭形和多角形 ;至培养第 9天DC细胞表面标志CD11c、CD80、CD86、MHCⅡ阳性率分别为 86 .32± 12 .14 %、76 .4 2± 8.4 5%、77.12± 9.0 5%、6 8.4 5± 6 .84 % ,IL 12浓度较未用LPS和TNF a组明显增加 (P <0 .0 1)。结论 :①结果所得细胞的形态和功能符合DC ;②用LPS和TNF a刺激可以获得成熟DC。

Isolation and Purification of Dendritic Cells From Mouse Bone Marrow and Their Expansion Culture Induced by Cytokines in Vitro

Objective:To isolate and purify dendritic cells(DCs) and their progenitors from the C57BL/C mouses bone marrow. Method:Bone marrow cell suspensions were prepared with no bacte ria and the erythrocytes were removed with red cell lysing buffer,the T and B ly mphocytes were removed with semi adhesion of DCs .DCs were induced in cu lture by both granulocyte macrophagecolony stimulating factor(GM-CSF) and inter leukin 4 (IL-4).The IL-12 and CD11c,CD80,CD86,MHCⅡwere assayed after LPS and TNF-a inducing DC 48 hours in the seventh day.Results:After 24～72 hours ,the DCs become relatively large,an d had availed appearance characteristic on fusiform,polyateral and finally stell ate.The positive rate of CD11c,CD80,CD86,MHCⅡ are 86.32±12.14%,76.42±8 .45%,77.12±9.05%,68.45±6.84%in DCs in the ninth day.The concentration of IL-12 is higher than that of the group without LPS and TNF-a inducing. Conclusion:The result shows that the cells form and the functio n seems DC,other way,the stimulation with LPS and TNF-a may get mature DC.