CIRCADIAN RHYTHM REPROGRAMMING DURING LUNG INFLAMMATION/ Microarray Experiment #2. Mus musculus

In this study we utilized a genome-wide approach to analyze circadian patterns of gene expression in mouse lung lungs, both in the basal state and in the setting of systemic inflammation caused by endotoxemia. We chose the lung because it represents a primary portal for systemic infection and organ failure in critically ill patients, and because the lung exhibits strong physiological circadian rhythms in health and in diseases such as asthma. The gene expression the data presented here was correlated to histological observations and metabolite measurements derived from the same biological samples, in order to obtain a broad picture of how inflammation impacts circadian rhythms in mouse lung. Overall design: To examine circadian regulation in mouse lung we performed 2 independent time-series experiments (Microarray Experiments #1 and #2), in which groups of mice were euthanized at 4 hour intervals for 48 hours. The purpose of Microarray Experiment #2 was to compare circadian gene expression in healthy lungs (samples labeled with the prefix “NT”) to lungs derived from endotoxemic animals (samples labeled with the prefix “LT”). For Microarray Experiment #2, 94 mice were placed under constant light conditions (LL 12:12, food ad libitum) at CT12 the prior day (7:00 PM local time). At CT10 (5:00 PM) on the day of the experiment a sub-group of 40 mice received a single intraperitoneal injection of 12 mg/kg E. coli O127:B8 endotoxin (LPS, Sigma L3129, Lot 029K4055), and sample collection began directly after. 3-4 mice per group were sacrificed at consecutive 4 hour intervals for 2-3 days. The left lung and the right upper lobe were frozen immediately in liquid nitrogen for microarray analysis. For mRNA isolation lung tissue was immersed in RNAlater (Qiagen) and total RNA was then extracted using the RNeasy Mini Kit (Qiagen). RNA quality (RIN>=7) was confirmed using an Agilent 2100 Bioanalyzer. RNA from all biological samples was labeled at once using the Ambion TotalPrep-96 RNA Amplification Kit. The samples were then blinded, randomized to chip position and hybridized to the Illumina MouseRef-8 v2.0 Expression BeadChips. For Microarray Experiment #2 at the Channing Division of Network Medicine (Brigham and Women’s Hospital). Two biological samples (NT00L1 and LT00L1) were represented by 2 technical replicates on the microarray. The remaining biological samples were represented by a single technical replicate.

本研究采用全基因组分析方法，探究小鼠肺部在基础状态以及内毒素血症诱发的全身性炎症状态下的基因表达昼夜节律模式。选择肺部作为研究对象，是因为其是重症患者发生全身性感染与器官衰竭的主要门户，且肺部在健康状态及哮喘等疾病中均存在显著的生理昼夜节律。本次公开的基因表达数据与同一份生物样本的组织学观察结果及代谢物检测结果进行关联分析，以全面阐明炎症对小鼠肺部昼夜节律的影响机制。
实验整体设计：为探究小鼠肺部的昼夜节律调控机制，本研究开展了2项独立的时序实验（微阵列实验#1与#2），实验中按每4小时一次的间隔对小鼠进行安乐死，持续48小时。微阵列实验#2的目的是对比健康小鼠肺部（样本前缀标记为"NT"）与内毒素血症小鼠肺部（样本前缀标记为"LT"）的基因表达昼夜节律差异。
在微阵列实验#2中，94只小鼠于前一日的昼夜节律时间（Circadian Time, CT）12时刻（当地时间19:00）被置于持续光照条件下（LL 12:12，自由摄食）。在实验当日的CT10时刻（当地时间17:00），将40只小鼠作为亚组，单次腹腔注射12mg/kg的大肠杆菌O127:B8内毒素（LPS，Sigma L3129，批号029K4055），随后立即开始样本采集。每组每间隔4小时处死3-4只小鼠，持续2-3天。将左肺与右肺上叶立即置于液氮中冷冻，用于后续微阵列分析。
用于mRNA分离的肺组织先浸入RNAlater试剂（Qiagen公司），随后采用RNeasy Mini Kit（Qiagen公司）提取总RNA。采用Agilent 2100生物分析仪对RNA质量进行验证，确保RNA完整性指数（RNA Integrity Number, RIN）≥7。所有生物样本的RNA均采用Ambion TotalPrep-96 RNA扩增试剂盒统一进行标记。随后对样本进行盲法处理，随机分配至芯片位点，并与Illumina MouseRef-8 v2.0表达微珠芯片进行杂交。该微阵列实验#2于网络医学钱宁分部（布里格姆妇女医院）完成。2份生物样本（NT00L1与LT00L1）在微阵列实验中设置了2次技术重复，其余生物样本均仅设置1次技术重复。