Profiling of Differentially Expressed Genes Using Suppression Subtractive Hybridization in an Equine Model of Chronic Asthma

BackgroundGene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. ObjectiveTo identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. MethodsEleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay. ResultsHorses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways. ConclusionsPathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies.

背景：基因表达分析（gene expression analyses）用于探究与疾病相关的信号通路（signaling pathways）。在哮喘研究中，此类分析主要源自对轻中度哮喘患者及健康对照的支气管活检（bronchial biopsies）样本的检测。但受伦理考量限制，目前关于哮喘患者外周肺组织的转录组谱（transcriptome profile）信息仍较为匮乏。
目的：本研究旨在鉴定与患heaves（一种自然发生的哮喘样病症）的马的外周肺组织慢性炎症及重塑相关的基因。
方法：本研究纳入11匹成年马（6匹患heaves，5匹健康对照），分别在临床缓解期（牧场饲养，Pasture）以及经30天圈养自然抗原刺激诱发疾病加重期（刺激期，Challenge）开展实验。于两个时间点均通过胸腔镜获取大块外周肺活检组织。采用抑制性消减杂交（suppression subtractive hybridization, SSH）技术，以临床疾病加重期患heaves马的肺cDNA为待检样本，分别消减健康对照（牧场饲养期与刺激期）及临床缓解期患马的肺cDNA。通过定量PCR（quantitative PCR）验证候选目的基因的差异表达情况。
结果：经抗原刺激后，患heaves的马可出现气道阻塞，健康对照马则无此表现。从消减文库中分离得到950个cDNA克隆，经斑点印迹阵列（dot blot array）筛选后，对其中224个表达差异最显著的克隆进行测序。在选取的22个基因中，15个的差异表达模式经定量PCR验证得到确认。在消减cDNA文库中，我们鉴定出了新型基因以及已有研究明确其在哮喘中功能的基因。其中与哮喘气道炎症及重塑密切相关的候选基因涉及PPP3CB/NFAT、RhoA以及LTB4/GPR44信号通路。
结论：本研究鉴定出了可作为哮喘抗炎及抗重塑治疗新靶点的信号通路。同时，本研究发现的既往与哮喘相关的基因，验证了该马类疾病模型可用于基因表达相关研究。