Isolation of Differentially Expressed Genes in Rat Hippocampus after Transient Global Cerebral Ischemia Using PCR mediating Subtractive cDNA Cloning.

为了阐明短暂性脑缺血后海马区现象的分子机制，我们利用聚合酶链式反应介导的消减克隆技术分离了短暂性脑缺血模型大鼠海马区差异表达基因。对数据库(日本DNA数据库)的同源性搜索表明，在我们分离的100多个片段中，多达1/2的片段是完全相同的，残基与已知的上载序列具有同源性，只有两个片段没有同源性。在鉴定的基因中，随机选择10个克隆，进行定量RT-PCR分析。其中一半(5110)在缺血后24小时内表达增加，是假手术的两倍以上。它们分别被鉴定为呋喃、丙皂苷、中性和碱性氨基酸转运体(NBAT)突触素IV和热休克蛋白105。进一步分析这些基因的克隆表达模式，发现Furin在缺血后6h达到高峰，而其他4个基因的峰值延迟，在缺血后24h出现。本研究还表明，利用PCR技术进行大鼠全脑缺血模型的差异表达基因克隆是一种非常有效和有用的工具。

To clarify molecular mechanisms underlying phenomenone in hippocampus after transient cerebral global isehemia, we have isolated differentially expressed genes in rat hippocampus of in vivo transient cerebral global ischemia model using PCR-mediating subtractive cloning. Homology search against database (DNA Data Bank of Japan) revealed that among over 100 fragments which we isolated, up-to 1/2 of these were identical, remains showed homology to known sequences up-loaded and only two displayed no homology. Among identified genes, 10 clones were selected randomly and quantitative RT-PCR analyses were performed. Half of them (5110) were revealed increased expression in 24 hour after ischemia over two times compared to sham operated. These are identified to furin, prosaposin, neutral and basic amino acid transporter (NBAT) synaptotagmin IV and heat shock protein 105, respectively. Furthermore, clonological expression pattern of mRNA of these genes were analyzed and it was revealed that furin had high peak at 6 hr after ischemia but peak of other 4 genes were delayed and had at 24 hr after. It was also suggested that PCR-mediating subtractive cDNA cloning is a very efficient and useful tool for analyzing genes in rat ischemic hippocampus of in vivo transient cerebral global ischemia model.