Comparative gene expression profile of 1,25-dihydroxyvitamin D3-treated human monocyte-derived dendritic cells

We have carried out global gene expression analysis to clarify the interrelationship between 1,25-dihydroxyvitamin D3 and differentiation-driven gene expression patterns in developing human monocyte-derived dendritic cells. Monocytes were treated with 10 nM 1,25-dihydroxyvitamin D3 or vehicle 14 hours after plating for 12 hours or 5 days. Monocytes, differentiating dendritic cells (+/-1,25-dihydroxyvitamin D3 for 12 hours) and immature dendritic cells (+/-1,25-dihydroxyvitamin D3 for 5 days) were harvested. This design allows one to identify genes regulated by differentiation and/or 1,25-dihydroxyvitamin D3 in human monocyte-derived dendritic cells. Human monocytes were obtained from buffy coats from healthy donors by Ficoll gradient centrifugation followed by immunomagnetic cell separation with anti-CD14-conjugated microbeads. Monocytes were cultured in RPMI-1640 supplemented with 10% FBS, 800 U/ml GM-CSF and 500 U/ml IL-4. Monocytes were treated with 10 nM 1,25-dihydroxyvitamin D3 or vehicle 14 hours after plating for 12 hours or 5 days. Monocytes, differentiating dendritic cells (+/-1,25-dihydroxyvitamin D3 for 12 hours) and immature dendritic cells (+/-1,25-dihydroxyvitamin D3 for 5 days) were harvested. Experiments were performed in biological triplicates representing samples from different donors. 15 samples were processed and hybridized to Human Genome U133 Plus 2.0 Arrays.

本研究开展全局基因表达分析（global gene expression analysis），以阐明1,25-二羟基维生素D3（1,25-dihydroxyvitamin D3）与发育中人单核细胞衍生树突状细胞（monocyte-derived dendritic cells）的分化驱动基因表达模式之间的相互关系。将单核细胞在贴壁培养14小时后，分别用10 nM的1,25-二羟基维生素D3或溶剂对照处理12小时或5天；随后收集单核细胞、分化中的树突状细胞（经1,25-二羟基维生素D3或对照处理12小时）以及未成熟树突状细胞（经1,25-二羟基维生素D3或对照处理5天）。该实验设计可用于鉴定人单核细胞衍生树突状细胞中受分化过程和/或1,25-二羟基维生素D3调控的基因。人单核细胞取自健康供者的血沉棕黄层（buffy coats），经Ficoll梯度离心（Ficoll gradient centrifugation）分离后，使用抗CD14偶联磁珠（anti-CD14-conjugated microbeads）进行免疫磁珠细胞分选获得。单核细胞于添加了10%胎牛血清（FBS）、800 U/ml粒细胞-巨噬细胞集落刺激因子（GM-CSF）及500 U/ml白细胞介素-4（IL-4）的RPMI-1640培养基中培养。单核细胞在贴壁培养14小时后，分别用10 nM的1,25-二羟基维生素D3或溶剂对照处理12小时或5天；随后收集单核细胞、分化中的树突状细胞（经1,25-二羟基维生素D3或对照处理12小时）以及未成熟树突状细胞（经1,25-二羟基维生素D3或对照处理5天）。实验设置生物学重复三次，每个重复样本取自不同供者。共制备15份样本，并将其与人类基因组U133 Plus 2.0芯片（Human Genome U133 Plus 2.0 Arrays）进行杂交。