Dysregulation of Macrophage Activation Profiles by Engineered Nanoparticles

To investigate how the phenotype of macrophages that have engulfed engineered nanoparticles (ENPs) differs from normal macrophages, we conducted Affymetrix microarray studies to identify the gene regulatory pathways affected by the ENPs. To mimic potential occupational exposure scenarios, the experimental design involved pretreatment of mouse primary bone marrow macrophages with the ENPs (25 mg/ml) for 24 hr, followed by removal of residual ENPs and challenging the macrophages with the TLR4 ligand and surrogate bacterial stimulus, lipopolysachharide (LPS) for 4 hr. The 4 hr challenge time was chosen based on preliminary studies which showed many of the proinflammatory gene expression responses peak between 2-6 hr after LPS treatment. Transcriptional responses were measured by global microarray analysis of mouse primary bone marrow macrophage cells. Groups (N=3 replicates) of primary mouse bone marrow macrophages were pretreated with selected engineered nanoparticless (33 nm iron oxide(SPIO) or 50 nm amorphous silica) at concentrations of 25 mg/ml for 24 hours. After removing the nanoparticles, macrophages were challenged with fresh media containing 10ng/ml lipopolysaccharide (LPS).

为探究吞噬工程化纳米颗粒（engineered nanoparticles，ENPs）的巨噬细胞（macrophages）表型与正常巨噬细胞的差异，我们开展了Affymetrix基因芯片实验，以识别ENPs所影响的基因调控通路。为模拟潜在的职业暴露场景，本实验设计为：以25 mg/ml的ENPs预处理小鼠原代骨髓巨噬细胞24小时，随后移除残留的ENPs，再使用Toll样受体4（TLR4）配体、模拟细菌刺激物脂多糖（lipopolysaccharide，LPS）对巨噬细胞进行4小时刺激。选择4小时刺激时长的依据为预实验结果：脂多糖处理后，多数促炎基因的表达应答在2~6小时区间内达到峰值。我们通过全基因组芯片分析检测了小鼠原代骨髓巨噬细胞的转录应答情况。本实验设置3组生物学重复（N=3），将原代小鼠骨髓巨噬细胞分别以25 mg/ml浓度的选定工程化纳米颗粒——33 nm氧化铁（SPIO）或50 nm无定形二氧化硅——预处理24小时。移除纳米颗粒后，巨噬细胞将被置于含有10 ng/ml脂多糖（LPS）的新鲜培养基中进行刺激。