Development of gene expression for FMDV-infected RAW264.7 cell. Mus

To determine whether type O FMDV-infected macrophage can release a variety of cytokines and activate innate immune responses, we have taken systematic functional genomics studies using microarray technology on differences expression gene of FMDV-infected and FMDV-uninfected mouse monocyte macrophage (RAW264.7 cells). We examined the expression patterns of 39,430 mouse genes probes, using Agilent mouse gene expression microarray. The results showed that 2541 genes were differentially expressed after FMDV infection, with 1207 up-regulated and 1334 down-regulated. Based on our previous data, the differential regulation of genes most associate with innate immune response. Overall design: The mouse monocyte macrophage cell line RAW264.7 and BHK-21 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. O type FMDV was grown in BHK-21 cells for 8h at 37°C. The virus titers were detected using the Reed-Muench method to determine the 50% tissue culture infective dose. The supernatants of infected cells were clarified and stored at −80°C. RAW264.7 cells were infected or uninfected with O type FMDV at MOI of 2 for 1h at 37°C, and then removed the supernatant, washed with phosphate-buffered saline (PBS) (pH 7.4), and cultured with complete DMEM for 8h at 37°C.

为探究O型口蹄疫病毒（Foot-and-Mouth Disease Virus, FMDV）感染的巨噬细胞能否释放多种细胞因子并激活天然免疫应答，本研究针对FMDV感染与未感染的小鼠单核细胞巨噬细胞（RAW264.7细胞）的差异表达基因，采用微阵列技术开展了系统性功能基因组学研究。本研究使用安捷伦（Agilent）小鼠基因表达微阵列，对39430个小鼠基因探针的表达模式进行了检测。结果显示，FMDV感染后共有2541个基因出现差异表达，其中1207个基因上调，1334个基因下调。结合前期实验数据，此类差异调控基因与天然免疫应答的关联最为紧密。

实验整体设计如下：将小鼠单核细胞巨噬细胞系RAW264.7与幼仓鼠肾细胞（Baby Hamster Kidney-21, BHK-21）接种于添加10%胎牛血清的达尔伯克改良伊格尔培养基（Dulbecco’s Modified Eagle Medium, DMEM）中培养。O型FMDV在BHK-21细胞中于37℃扩增培养8小时。采用瑞德-门奇（Reed-Muench）法检测病毒滴度，以确定50%组织细胞感染量（TCID₅₀）。收集感染细胞的上清液并进行澄清处理后，于-80℃低温保存。以感染复数（Multiplicity of Infection, MOI）为2的感染比例，用O型FMDV感染RAW264.7细胞，于37℃吸附1小时后移除上清液，采用pH7.4的磷酸盐缓冲液（Phosphate Buffered Saline, PBS）洗涤细胞，随后添加完全DMEM培养基，于37℃继续培养8小时。