基因芯片筛查亚溶解型C5b-9刺激大鼠肾小球系膜细胞基因表达差异的研究

目的:应用高通量的基因芯片技术筛查亚溶解型C5b-9(sublytic C5b-9)介导大鼠肾小球系膜细胞(glomerular mesangial cells, GMCs)的基因表达谱的变化?方法:体外分离?培养大鼠GMCs,人工质控sublytic C5b-9复合物的形成,并用其刺激培养GMCs,然后分别提取sublytic C5b-9刺激GMCs 40 min?3 h及未刺激对照组细胞的总RNA?经逆转录合成生物素标记的cDNA探针,然后与基因芯片杂交(涵盖31 000个转录本,代表28 000个基因)?经Affymetrix Scanner 3000 扫描芯片荧光信号图像?GCOS1.4读取数据后将差异表达基因注释于GO基因功能分类体系,分析其相应的功能?结果:芯片检查数据显示,sublytic C5b-9刺激GMCs 40 min 时上调2倍以上及下调2倍以上的基因分别为4 124个和2 234个,刺激3 h时上调2倍以上及下调2倍以上的基因分别为4 512个和2 859个;而sublytic C5b-9刺激GMCs 40 min和3 h后同时上调2倍以上及下调2倍以上的基因分别为2 325个和1 419个?sublytic C5b-9刺激GMCs表达差异基因其功能主要涉及生物学过程调节?生物调节?定位相关?刺激应答?生长发育?增殖?代谢过程?细胞生理过程?多细胞机体过程等?结论:Sublytic C5b-9 刺激GMCs确能引起基因表达谱的变化?

The changes of gene expressed differences in rat glomerular mesangial cells with sublytic C5b-9 attack by gene chip

Objective:To screen differentially expressed genes in rat glomerular mesangial cells(GMCs)in response to sublytic C5b-9 by microarray of high flux. Methods:Rat primary GMCs were cultured and stimulated with or without sublytic C5b-9, then the total RNA in GMCs was extracted,and the RNA was reversely transcribed to biotinylated labeled cDNA probes. The mixed probes were hybridized for cDNA microarray. Then, the chips were subsequently scanned by Affymetrix Scanner 3000 and read with software GCOS1.4. Then differentially expressed genes were identified and annotated to Gene Ontology(GO)database in order to analyze these genes function. Results:The results showed that among expression profile of 31 000 genes, up-regulated and down-regulated genes over 2 folds under sublytic C5b-9 attacking for 40 min were 4 124 and 2 234, and were 4 512 and 2 859 under attacking for 3 h respectively. Meanwhile, there were 2 325 genes up-regulated and 1 419 genes down-regulated over 2 folds under sublytic C5b-9 attacking for 40 min or 3 h. Analysis of these differences of genes function by GO, these differentially expressed genes function mainly involved in regulation of biological process, biological regulation, localization,response to stimulus, developmental process,proliferation, metabolic process,cellular process, multicellular organismal process and so on. Conclusion:sublytic C5b-9 stimulating rat GMCs indeed could cause the changes of gene expression.