人外周血树突状细胞体外诱导培养及表型鉴定

目的从健康人外周血中分离单个核细胞（peripheral blood mononuclearcell,PBMC）,经体外诱导培养获取成熟树突状细胞（dendritic cell,DC）.方法取健康人新鲜外周血,经密度梯度离心法分离,获得单个核细胞,在含10%胎牛血清的RPMI-1640培养基中培养.置37℃、5%CO2培养箱内静置培养2 h后除去悬浮细胞.培养液中加入rhGM-CSF和rhIL-4继续培养贴壁细胞5 d,然后加入TNF-α促进其分化成熟.倒置显微镜下观察DC形态,流式细胞仪（flowcytometer,FCM）对其进行表型鉴定.结果显微镜下观察DC细胞形态不规则,表面有大量的毛刺状突起;成熟DC细胞表面CD83、CD80分子表达明显增高.结论用rhGM-CSF、rhIL-4和TNF-α联合培养可以从健康人外周血诱导培养出成熟的树突状细胞.

Inducing Culture in vitro and Phenotypic identification of Dendritic Cells from Peripheral Blood of Healthy People

Objective To isolate the PBMC from peripheral blood of healthy human,and get mature dendritic cell（DC）by inducing culture in vitro.Methods PBMCs were isolated from fresh peripheral blood of healthy people by the method of density-gradient centrifugation.The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum at 37 ℃ in a 5% CO2 humidified atmosphere for 2 hours,and then the suspended cells were removed.Then recombinant human granulocyte-macrophage colony-stimulating factor（rhGM-CSF）and recombinant human interleukin-4（rhIL-4）were added into medium,and the adherent cells were cultured to continue for 5 d.Tumor necrosis factor-α（TNF-α）was added to promote DCs＇maturation and differentiation.Then the morphological features were observed with inverted microscope and the phenotypes of cells were detected by flow cytometer（FCM）.Results Under the microscope,the dendritic cells were in irregular shape with slender synapses on their surface.The expressions of CD83 and CD80 on mature dendritic cells were significantly higher on 8d than that on 4d.Conclusion Mature dendritic cells can be cultured from peripheral blood of healthy people in vitro by utilizing rhGM-CSF,rhIL-4 and TNF-α