人类外周血来源树突状细胞的体外诱导培养

目的 探讨体外诱导培养较高纯度和成熟度的树突状细胞(DC)的方法.方法 密度梯度离心分离人类外周血单核细胞,直接贴壁法收集前体细胞,含人类血清及细胞因子rhGM-CSF和rhIL-4完全RPMI1640培养基,37 ℃、5%CO2孵育培养;第3天全量换液并添加细胞因子,第5天加入rhTNF-α促进成熟,第8天收获悬浮细胞.同时进行形态学和细胞表型分析.结果 镜下表现为典型的DC形态特征;流式分析细胞表型CD1a阳性表达从第5天的(18.69±8.73)%增加到第8天的(78.07±9.43)%,CD83表达从(14.74±4.06)%增加到(46.82±14.15)%,第5天CD80表达(9.82±4.61)%,第8天CD80表达(60.11±20.50)%;40 ml外周血约得(3.12±1.30)×106个DC.结论 该方法可以体外诱导培养出较大数量和较高纯度的成熟DC,并且培养体系成熟,操作性和可重复性强.

Induction and culturing of dendritic cells derived from human peripheral blood monocytes in vitro

Objective To generate high purity and maturity DC from human peripheral blood monocytes in vitro.Methods PBMC were isolated directly from whole blood by density gradient centrifugation and purified by collecting the attached cell,DC were then generated by induction and culturing PBMC for five days with RPMI1640 medium containing rhGM-CSF and rhIL-4 in vitro,and under the condition of 37 ℃,5% CO2.On the fifth day,rhTNF-α was added into DCs cultures,which were then incubated for three additional days.The morphology was monitored by light microscopy,and the phenotypes were determined by FCM.Results After eight days of culture,the cells developed typical and significant dendritic morphology and plenty of cells expressed CD1a, CD80 and CD83,features of DC.Including(78.07±9.43)%CD1a,(60.11±20.50)% CD80 and(46.82±14.15)% CD83 were expressed.About (3.12±1.30)x106 DC cells were derived from 40ml human peripheral blood.Conclusion The way to generate DCs is simple and easy.The DCs produced by this method acquired high purity and maturity antigenic characteristics of DCs.