Xie Lifang, Pandey Ritu, Xu Beibei, Tsaprailis George, Chen Qin M
Department of Pharmacology, College of Medicine, University of Arizona, 1501 N. Campbell Ave., Tucson, AZ 85724, USA.
Biogerontology. 2009 Apr;10(2):125-51. doi: 10.1007/s10522-008-9157-3. Epub 2008 Jul 25.
A number of lines of evidence suggest that senescence of normal human diploid fibroblasts (HDFs) in culture is relevant to the process of aging in vivo. Using normal human skin diploid fibroblasts, we examine the changes in genes and proteins following treatment with a mild dose of H2O2, which induces premature senescence. Multidimensional Protein Identification Technology (MudPIT) in combination with mass spectrometry analyses of whole cell lysates from HDFs detected 65 proteins in control group, 48 proteins in H2O2-treated cells and 109 proteins common in both groups. In contrast, cDNA microarray analyses show 173 genes up-regulated and 179 genes down-regulated upon H2O2 treatment. Both MudPIT and cDNA microarray analyses indicate that H2O2 treatment caused elevated levels of thioredoxin reductase 1. Semi-quantitative RT-PCR and Western-blot were able to verify the finding. Out of a large number of genes or proteins detected, only a small fraction shows the overlap between the outcomes of microarray versus proteomics. The low overlap suggests the importance of considering proteins instead of transcripts when investigating the gene expression profile altered by oxidative stress.
多项证据表明，培养的正常人二倍体成纤维细胞（HDFs）的衰老与体内衰老过程相关。我们使用正常人皮肤二倍体成纤维细胞，研究了用低剂量H2O2处理后基因和蛋白质的变化，H2O2可诱导过早衰老。多维蛋白质鉴定技术（MudPIT）结合对HDFs全细胞裂解物的质谱分析，在对照组中检测到65种蛋白质，在H2O2处理的细胞中检测到48种蛋白质，两组共有109种蛋白质。相比之下，cDNA微阵列分析显示，H2O2处理后有173个基因上调，179个基因下调。MudPIT和cDNA微阵列分析均表明，H2O2处理导致硫氧还蛋白还原酶1水平升高。半定量RT-PCR和蛋白质印迹能够验证这一发现。在检测到的大量基因或蛋白质中，只有一小部分在微阵列与蛋白质组学结果之间存在重叠。低重叠率表明，在研究氧化应激改变的基因表达谱时，考虑蛋白质而非转录本具有重要意义。
