Analysis of disease-related genes by using microaaray DNA chip with the normalized cDNA library

大规模的基因表达监测是阐明细胞事件的有力手段。DNA微阵列技术使我们能够识别全基因组的表达谱，并给生物学研究带来深刻的影响，如药理学。该技术还可应用于药物发现和疾病的分子分类。我们引入了微阵列来发现与疾病状态相关的基因的新功能。在分析动物疾病模型的组织时，我们首先尝试使用从Unigene克隆(研究遗传学)中随机选择的cDNA的微阵列，并发现很少有克隆成功杂交，可能是因为组织特异性基因表达。为了解决这个问题，我们的目标是利用待分析组织的cDNA文库来制作微阵列；然而，由于细胞中存在冗余的mRNA物种，普通的cDNA文库包含了高频率的不希望看到的克隆。消除普通cDNALibra…中的冗余更多(“归一化”)，我们选择了“减法文库”和“命中挑选”两种方法。“消减文库”方法是一种传统的方法，而“命中-挑选”方法是我们将宏阵列和机器人系统相结合而发展起来的一种新方法；后一种方法被称为“击中-挑选”，因为我们通过过滤器杂交选择后收集到了理想的克隆。应用DNA芯片和归一化的cDNA文库，对IgA肾病模型动物进行分析。免疫球蛋白A肾病(IgAN)是世界上最常见的原发肾小球疾病，其分子机制尚不清楚。HIGA(高血清IgA)小鼠是一种有效的IgA肾病模型，几乎具有包括系膜细胞增殖在内的所有病理特征。在这里，我们通过对病变肾脏的基因芯片分析，阐明了与IgAN相关的基因表达模式。特别是，我们发现了几个调控细胞周期和增殖的基因的表达增强，包括生长因子及其受体，以及1-磷酸鞘氨醇(SPP)的受体-内皮分化基因-5(EDG5)。作为一种生长因子，血小板衍生生长因子(PDGF)可诱导系膜细胞增殖过程中EDG5的表达显著上调，并与SPP协同促进细胞增殖。基因组学方法使我们能够确定一个过程中涉及的基因家族，并可以表明增强的PDGF-EDG5信号在IgAN的进展中起着重要作用。较少

Large scale monitoring of gene expression is a powerful approach to clarify the cellular events. DNA microarray technologies permit us to recognize genome-wide expression pro filing, and bring a profound impact to biological research, such as pharmacology. This technology can also be applied to drug discovery and molecular classification of diseases. We introduced microarray to discover novel function of genes involved in disease states. In analyzing tissues of an animal disease model, we first attempted to use microarrays with cDNAs randomly selected from Unigene clones (Research Genetics), and found that few clones successfully hybridized presumably because of the tissue-specific gene expression. To resolve this problem, we aimed to fabricate microarrays with cDNA library of the tissue to be analyzed ; however, ordinary cDNA libraries contain a high frequency of undesirable clones because of redundancy of mRNA species in the cell. To get rid of the redundancy from ordinary cDNA libra … More ries ("normalization"), we chose two approaches of "subtractive library" and "hit-picking". "Subtractive library" approach is a conventional method, while "hit-picking" approach is a novel one we developed by combining macroarray and robotic systems ; we named the latter method "hit-picking" since we collect desirable clones after selection by filter hybridization. By using DNA chip with normalized cDNA library, we analyzed IgA nephropathy model animal. The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression asosociated with IgAN by analyzing the diseased kidneys on cDNA microarrays. In particular, we showed an enhanced expression of several genes regulating the cell cycle and proliferation, including growth factors and their receptors, as well as endothelial differentiation gene-5 (EDG5), a receptor for sphingosine 1-phosphate (SPP). One of growth factors, platelet-derived growth factor (PDGF) induces a marked upregulation of EDG5 in proliferative mesangial cells, and promotes cell proliferation synergistically with SPP. The genomic approach allows us to identify families of genes involved in a process, and can indicate that an enhanced PDGF-EDG5 signaling plays an important role in the progression of IgAN. Less