哮喘患儿外周血单个核细胞全基因组表达谱的差异研究

Profiling of differential expression in asthma and healthy children’s peripheral blood mononuclear cells by whole-genome microarray

目的:筛选哮喘患儿与对照组儿童外周血的基因表达谱差异,寻找与哮喘防治相关的靶基因。方法:从5名哮喘患儿和5名对照组儿童外周血单个核细胞中提取总RNA,利用全基因组表达谱芯片进行检测,选取经非配对t检验计算所得P≤0.05、同时基因表达变化≥2倍的差异表达基因,以荧光定量PCR(qRT-PCR)验证芯片结果。通过生物信息学软件对初步筛选的差异基因进行层次聚类分析和Gene Ontology(GO)功能分类分析。结果:从45 033条表达基因谱中筛选出哮喘患儿与对照组儿童差异表达2倍以上且P≤0.05的已命名基因758个(含上调基因345个,下调基因413个),其GO生物学过程功能分类主要涉及免疫反应、对外部刺激的反应、信号转导及分子功能的负性调节、细胞死亡、凋亡及其调节等。其中有29个基因与哮喘、气道炎症或气道重构有关,且变化趋势与文献报道一致(含上调基因14个,下调基因15个),并可被层次聚类分析划分为9类。qRT-PCR验证结果与芯片结果一致。结论:用全基因组表达谱芯片可以筛选出哮喘患儿与对照组儿童外周血单个核细胞中的差异表达基因,进一步的数据挖掘很可能寻找到与哮喘防治相关的靶基因或靶通路。

AIM： To compare the differences of whole-genome expression in peripheral blood mononuclear cells（PBMC） between asthma children and healthy controls.METHODS： The subjects were 5 cases of asthma children and 5 cases of healthy controls.Total RNA was extracted from PBMC and subjected to microarray analysis with NimbleGen human gene expression array.Unpaired t-test algorithm was used to screen the differentially expressed genes when P≤0.05 and fold change ≥ 2.Real-time quantitative PCR（qRT-PCR） was performed to verify the microarray results.Classification and function of the differential genes were illustrated by bioinformatic processing including hierarchical clustering and Gene Ontology analysis.RESULTS： According to the fold change ≥ 2 and P≤0.05,there were 758 named genes（345 up-regulated and 413 down-regulated） out of 45 033 analyzed transcripts were differentially expressed in PBMC of the asthma children compared with the control children.They were mostly related to immune response,response to stress and stimulus,negative regulation of signal transduction and molecular function,and regulation of cell death and apoptosis.Among these transcripts,29 genes（14 up-regulated and 15 down-regulated） related to asthma,airway inflammation or remodeling could be classified into 9 categories and their variation tendency was consistent with the previously reported data.The results of qRT-PCR successfully confirmed the data of microarray.CONCLUSION： Whole-genome microarray can screen the differentially expressed genes in PBMC between the asthma children and the healthy controls.Further data mining is required to trace target genes or target pathways related to prevention and treatment of asthma.