Objective： This study is aimed at developing a simple and easy way to generate dendritic cells （DCs） from human peripheral blood monocytes （PBMCs） in vitro. Methods： PBMCs were isolated directly from white blood...Objective： This study is aimed at developing a simple and easy way to generate dendritic cells （DCs） from human peripheral blood monocytes （PBMCs） in vitro. Methods： PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods （collecting the attached cell and removing the suspension cell）. DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor （rhGM-CSF） and 20 ng/ml recombinant human interleukin-4 （rhIL-4） in vitro. On the sixth day, TNF-alpha （TNFα） 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions （MLR） were used to characterize DC function after TNFα or lipopolysaccharide （LPS） stimulations for 24 h. Results： After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CDIa, CD80 and CD86, features of DCs. TNFα treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. Conclusion： This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.展开更多
文摘Objective： This study is aimed at developing a simple and easy way to generate dendritic cells （DCs） from human peripheral blood monocytes （PBMCs） in vitro. Methods： PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods （collecting the attached cell and removing the suspension cell）. DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor （rhGM-CSF） and 20 ng/ml recombinant human interleukin-4 （rhIL-4） in vitro. On the sixth day, TNF-alpha （TNFα） 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions （MLR） were used to characterize DC function after TNFα or lipopolysaccharide （LPS） stimulations for 24 h. Results： After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CDIa, CD80 and CD86, features of DCs. TNFα treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. Conclusion： This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.
