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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 mesan-gial 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。
OBJECTIVE: To screen the gene expression profiling of glomerular mesang-gial cells (GMCs) sub-lytically C5b-9 mediated by high-throughput microarray. Methods: The GMCs were isolated and cultured in vitro. The sublytic C5b-9 complex was artificially controlled and cultured. GMCs were stimulated with sublytic C5b-9 for 24 and 30 minutes, respectively. RNA. Biotinylated cDNA probes were synthesized by reverse transcription and then hybridized to the gene chip (covering 31 000 transcripts representing 28 000 genes). The fluorescence signal of the chip was scanned by Affymetrix Scanner 3000. After the data was read by GCOS1.4, the differentially expressed genes were annotated with GO gene function classification system to analyze their corresponding functions. Results: According to the chip test data, 4 124 and 2 234 genes were up-regulated and sub-activated at 40 min after sublytic C5b-9 stimulation for 40 min, respectively, up-regulated more than 2-fold and down-regulated more than 2-fold at 3 h Of the genes were 4 512 and 2 859 respectively, while those of sublytic C5b-9 stimulated GMCs to up-regulate more than 2-fold and down-regulated more than 2-fold at 40 min and 3 h, respectively, to be 2 325 and 1 419, respectively. The function of sublytic C5b-9 to stimulate GMCs to express differentially expressed genes mainly involves biological process regulation, biological regulation, localization related, stimulation response, growth and development, proliferation, metabolic process, cell physiology process and multicellular body process. CONCLUSIONS: Sublytic C5b-9 stimulates GMCs indeed to cause changes in gene expression profiles.