Expression data from hippocampus in mouse brain

Our microarray results showed there were up-regulated 28 genes and down-regulated 29 genes, which related depression and inflammatory response such as cytokine-cytokine receptor interaction, chemokine signaling pathway, dopaminergic synapse, glutamatergic synapse, GABAergic synapse, cholinergic synapse, and serotonergic synapse. Among these genes, especially, higher hippocampal mRNA expression of transthyretin (Ttr), Zinc finger protein of the cerebellum 1 (Zic1), and Ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) was found in kososan-administered defeated mice than water-administered defeated mice DNA microarray analysis was conducted using isolated RNAs extracted from the hippocampus as described previously (Sasaki et al., 2017). Double-stranded cDNA was synthesized from 100 ng of total RNA with the GeneAtlas 3' IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). Biotin-labeled amplified RNA (aRNA) was synthesized using the GeneChip 3´ IVT Express Kit (Affymetrix Inc.). Purified aRNA was fragmented using the GeneAtlas 3´ IVT Express Kit and hybridized for 16 h at 45 °C using the GeneChip Mouse Genome 430 PM microarray (Affymetrix Inc.). The chip was washed and stained in the Gene Atlas Fluidics Station 400 (Affymetrix Inc.), and the resulting image was scanned using the GeneAtlas Imaging Station (Affymetrix Inc.). Data analysis was performed using the Affymetrix expression console (http://www.affymetrix.com) and the online data tool DAVID (Database for Annotation, Visualization and Integrated Discovery, v6.8, National Institute of Allergy and Infectious Diseases, NIH, USA, https://david.ncifcrf.gov/) (Huang et al., 2007a; Huang et al., 2007b). Among the clustered genes, the genes that contain the gene ontology concerning depression and inflammatory response (e.g., cytokine-cytokine receptor interaction, chemokine signaling pathway, dopaminergic synapse, glutamatergic synapse, GABAergic synapse, cholinergic synapse, and serotonergic synapse) were selected using DAVID tool and manually.

本研究的基因芯片（microarray）结果显示，共筛选得到28个上调基因与29个下调基因，这些基因与抑郁症及炎症反应密切相关，涉及的通路包括细胞因子-细胞因子受体相互作用（cytokine-cytokine receptor interaction）、趋化因子信号通路（chemokine signaling pathway）、多巴胺能突触（dopaminergic synapse）、谷氨酸能突触（glutamatergic synapse）、γ-氨基丁酸能突触（GABAergic synapse）、胆碱能突触（cholinergic synapse）以及5-羟色胺能突触（serotonergic synapse）。其中，给予kosan的受挫小鼠的海马体中转甲状腺素蛋白（transthyretin, Ttr）、小脑锌指蛋白1（Zinc finger protein of the cerebellum 1, Zic1）以及外核苷酸焦磷酸酶/磷酸二酯酶2（Ectonucleotide pyrophosphatase/phosphodiesterase 2, Enpp2）的mRNA表达水平，显著高于给予饮水的受挫对照组小鼠。本研究参照此前报道的方法（Sasaki等，2017），从海马体中提取总RNA并开展基因芯片分析：以100 ng总RNA为模板，使用GeneAtlas 3'端体外转录快速表达试剂盒（美国加利福尼亚州圣克拉拉市Affymetrix公司）合成双链cDNA；随后使用GeneChip 3'端体外转录快速表达试剂盒（Affymetrix公司）合成生物素标记的扩增RNA（amplified RNA, aRNA）。经纯化的aRNA使用GeneAtlas 3'端体外转录快速表达试剂盒进行片段化处理，再使用GeneChip小鼠基因组430 PM基因芯片（Affymetrix公司）在45℃下杂交16小时。将芯片置于Gene Atlas流体工作站400（Affymetrix公司）中完成洗涤与染色，之后使用GeneAtlas成像工作站（Affymetrix公司）扫描获取芯片图像。数据分析采用Affymetrix表达控制台（Affymetrix expression console，http://www.affymetrix.com）与在线数据分析工具DAVID（注释、可视化与集成发现数据库（Database for Annotation, Visualization and Integrated Discovery, v6.8），美国国立卫生研究院过敏与传染病研究所，https://david.ncifcrf.gov/）（Huang等，2007a；Huang等，2007b）完成。在聚类得到的基因集中，通过DAVID工具与人工筛选的方式，选出与抑郁症及炎症反应相关的基因本体（gene ontology, GO）条目对应的基因，涵盖前述各信号与突触通路。