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The analysis of genes related to the progressive kidney disease using gene expression progiles

使用基因表达谱分析与进行性肾病相关的基因

基本信息

批准号：
11671035
负责人：
TAKENAKA Masaru
金额：
$ 2.5万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The protein-overloaded proteinuira model has been reported to study renal damages caused by proteinuria, which we confirmed the same histological changes. We collected approximately 20cm mouse renal proximal tubules of disease model mice by micordissection methods under macroscopy, and extracted mRNA.The gene expression profiles were constructed by direct sequencing of 3' end of over 3000 cDNA clones that were randomly picked up from the transformants of un-amplified cDNA library. The profile contained the information about relative amount of mRNA expression. We compared those with gene expression profiles of normal tissues and cells. It revealed that the expression of genes related to proteolysis and inflammation were increased, whereas several abundant genes in normal proximal tubules were decreased, e.g. thymic shared antigen (TSA-1). Several genes were analyzed by Northern blot analysis, laser microdissection-real time PCR method, in situ hybridization and/or immunohistochemistry. It showed that mRNA expression of one of those genes, TSA-1, was increased in proximal tubular cells. Immunohistochemistry staining confirmed that its protein expression was a basolateral pattern and induced by proteinuria. The data of gene expression profile and microarray analysis revealed that osteopontin was induced by proteinuria in the kidney. When the informed consents were obtained, OPN mRNA expression in proximal tubules of renal biopsy specimen was quantified by the LMM method. The analyses revealed that OPN expression in proximal tubules was exponentially increased along with the amount of proteinuria.

蛋白尿模型已被报道用于研究蛋白尿引起的肾损害，我们证实了相同的组织学变化。本研究采用肉眼下显微解剖的方法收集了疾病模型小鼠近端肾小管约20 cm的组织，提取mRNA，从未扩增的cDNA文库中随机挑取3000多个cDNA克隆进行3'端直接测序，构建了基因表达谱。该谱包含关于mRNA表达的相对量的信息。我们将其与正常组织和细胞的基因表达谱进行了比较。结果表明，与蛋白水解和炎症相关的基因表达增加，而正常近端小管中的几个丰富的基因表达减少，如胸腺共享抗原（TSA-1）。采用北方杂交、激光显微切割-实时荧光PCR、原位杂交和/或免疫组织化学等方法对基因进行分析。结果表明，其中一个基因TSA-1的mRNA表达在近端肾小管细胞中增加。免疫组织化学染色证实其蛋白表达为基底外侧型，并由蛋白尿诱导。基因表达谱和芯片分析结果显示，骨桥蛋白在肾脏中的表达受蛋白尿的诱导。当获得知情同意后，通过LMM方法定量肾活检标本近端小管中OPN mRNA的表达。分析显示，近端小管中的OPN表达随着蛋白尿量的增加而沿着呈指数增加。