健康人外周血树突状细胞体外培养及表型鉴定

目的 分离人外周血单个核细胞,经体外诱导培养获取成熟树突状细胞(DC),并对树突状细胞表型表达鉴定,为进一步研究DC功能提供基础.方法 取健康成人新鲜外周血,经密度梯度离心法分离获得外周血单个核细胞,2 h贴壁后加入粒细胞集落刺激因子(GM-CSF)、白介素(IL)-4,第6天加入肿瘤坏死因子-α(TNF-α)刺激DC成熟,倒置显微镜观察每日细胞形态;分别于第1天、第6天、第8天用流式细胞仪对其进行表型鉴定;同种异体混合淋巴细胞反应观察对T细胞的抗原提呈能力 倒置显微镜下DC细胞形态不规则,表面有毛刺状突起,呈DC典型形态学特征;成熟DC细胞表面CD1a、CD80、CD83、人类白细胞抗原(HLA)-DR表达明显增加,混合淋巴细胞反应OD值增加.结论 用GM-CSF、IL-4和TNF-α可以诱导健康成人外周血单个核细胞,培养出成熟的DC细胞,为进一步研究DC功能提供基础.

In vitro culture and phenotypic identification of dendritic cells from healthy human peripheral blood

Objective To isolate peripheral blood mononuclear cell （PBMC） from healthy people, and induce, culture and phenotypic identificate dendritic cells （DC） from PBMC in vitro, in order to provide a basis for further study of DC function. Methods PBMC were sorted from fresh healthy human peripheral blood by density gradient centrifugation, cultured in the presence of GM - CSF and IL - 4 after two hours and TNF - ct after 6 days was added to promote DC maturation and differentiation. Then the morphological features were observed with inverted micro- scope and the phenotypic characterization was detected by flow cytometry （FCM） in the first day, the 6th day and the 8th day. Antigen - presen- ting ability was reflected from mixed lymphocyte reaction. Results The DC were in irregular shape with slender synapses on their surface. There were higher levels of CDla, CDSO, CD83 and HLA - DR on mature DC surface. In addition, there was a higher optical density （OD） value of mixed lymphocyte reaction in experimental group than that in control group. Conclusion Mature DC can be cultured from peripheral blood of healthy people in vitro by using cytokine GM - CSF, IL - 4, and TNF - cL. And the in vitro culture of DC provides the basis for further study of DC function.