应用基因芯片技术对系统性红斑狼疮患者外周血基因表达谱的初步研究

Analysis of gene expression profiles in peripheral blood of systemic lupus erythematosus using oligonucleotide DNA microarray

目的 研究系统性红斑狼疮 (SLE)患者与正常对照外周血的基因表达谱的改变 ,以探讨SLE发病机制、诊断及鉴别诊断 ,以及基因功能的研究。方法 首先从外周血提取总RNA ,反转录合成单链、双链cDNA后 ,体外转录合成生物素标记的cRNA与基因芯片进行杂交 ,再通过抗原抗体反应机制标记上荧光染料Cy3后 ,使用基因芯片扫描仪进行图像扫描。使用分析软件进行表达差异和聚类分析。结果 在 30 0 0多个基因点中 ,与正常对照相比较 ,鉴定出 94个基因存在表达差异相关基因 ,其中有 33个基因表达上调 ,6 1个基因表达下调。这些表达差异基因有细胞因子及受体、转录相关基因、凋亡相关基因、生长分化相关基因、信号转导相关基因、细胞周期相关基因、电子传递和氧化相关基因、转移酶相关基因、核酸酶相关基因等。聚类分析结果显示不同的疾病以及不同临床表现的同一疾病其外周血的基因表达谱是有差异的 ,可以用于疾病的诊断和鉴别诊断。基因聚类发现 ,根据表达谱聚类在一起的基因存在功能上的相似性 ,可以用来发现已知基因的免疫相关功能。结论 该基因芯片技术方法有较高的重复性和稳定性 ,能有效地进行SLE致病基因的筛选 ,更好地理解SLE发生的分子机制、诊断及鉴别诊断 ,以及基因功能的研究。

Objective To investigate pathogenesis,diagnosis and differential diagnosis of systemic lupus erythematosus (SLE),and find new gene function association with immunity through study on the difference in gene expression of peripheral blood white cells between SLE patients and healthy controls.Methods Total RNA was extracted from peripheral blood cells of normal subjects and SLE patients,and single and double stranded cDNA templates from total RNA.The transcription of cRNA probe labelled with biotin and hybridization of probe with microarray were performed.Binding of streptavidin to biotin and then amplification with first antibody was done.Further amplification with Cy3 conjugated second antibody and detection of Cy3 dye with ScanArray 5000 was accomplished.Through QuantArray microarray analysis software the scan imaging information was converted into numeric data,and normalization and clustering analysis with GeneSpring microarray analysis software were to find interesting genes.Results Data were analyzed to select genes which were over or under expressed in SLE patients versus controls.Among the 3 360 target genes,94 were identified in SLE patients that were either over or under expressed as compared to controls,33 were up regulated and 61 down regulated.These genes included genes associated with cytokine and receptors,with transcriptional regulation, with apoptosis and oncogenes,with differentiation and development,with signal transduction,with cell cycle or cyclins,with electron transport and oxidoreductase,with transferase,DNase and others.Clustering results showed different gene expression profiles of different diseases and different clinical manifestations of same disease,so this differential gene expression profiles could help to diagnose and differentially diagnose .Gene clustering results showed the co ordinate regulation of many functionally related genes based on the degree of similarityintheirexpressionprofilesand could be used to find the function of these known immunity related genes.Conclusion These results successfully demonstrate that the use of the oligonucleotide DNA microarray technique is effective for screening the differentially expressed genes of SLE,and understanding the pathogenesis of SLE all the better,it helps us diagnose and differentially diagnose,find the new gene function associated with immunity.