Microtranscriptomics and microproteomics of human glomerular diseas

人类肾小球疾病的微转录组学和微蛋白质组学

• 批准号: 17390247
• 负责人: YAMAMOTO Tadashi
• 金额: $ 10.37万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390247/
• 关键词: transcriptome; proteome; chronic kidney disease; gene; protein; mass spectrometry; database; glomerulus; 蛋白質; 二次元電気泳動; 質量分析; DNAアレー; 腎臓; 糸球体疾患

This project was aimed to establish platform for transcriptomics and proteomics of human kidney biopsy samples from kidney disease patients.1) Human glomerulus cDNA library was prepared and approximately 6,000 clones were sequenced. The normal human glomerular transcriptome was analyzed by DNA microarray technique and the database was created by annotating the glomerular genes. By the analysis several genes were demonstrated as those uniquely expressed in the glomerulus. Their expression was localized by in situ hybridization and immunohistochemistry techniques.2) The proteome of normal human glomerulus was analyzed by 2D gel electrophoresis and nanoLC-ESI TOF MS. More than 7000 proteins originated from about 3000 genes were identified in the glomerulus and a glomerular proteome database was created in association with immunohistochemistry image data provided from the Human Protein Atlas project of Human proteome organization (HUPO).3) To establish procedures for microtranscriptomics and microproteomics to analyze glomerulus in human kidney biopsy samples, glomerular sections were microdissected and mRNA and protein were isolated. From 50 glomerular sections, about one pg each of RNA and protein was isolated and was applicable for microanalysis. Contamination of human keratin and co-existence of human plasma proteins in the glomerular protein samples were also established.

本项目旨在建立肾脏病患者肾活检标本的转录组学和蛋白质组学研究平台。1）制备人肾小球cDNA文库，并对约6,000个克隆进行测序。应用基因芯片技术分析正常人肾小球转录组，并对肾小球基因进行注释，建立肾小球基因数据库。通过分析，几个基因被证明是那些独特的表达在肾小球。采用原位杂交和免疫组化技术对它们的表达进行定位。2）采用二维凝胶电泳和纳米LC-凝胶电泳技术对正常人肾小球蛋白质组进行分析。在肾小球中鉴定了来自约3000个基因的超过7000种蛋白质，并且与人类蛋白质图谱项目提供的免疫组织化学图像数据相关联地创建了肾小球蛋白质组数据库。3）为了建立用于微转录组学和微蛋白质组学分析人肾活检样品中肾小球的程序，显微切割肾小球切片并分离mRNA和蛋白质。从50个肾小球切片中分离出约1 pg的RNA和蛋白质，可用于微量分析。还确定了肾小球蛋白样品中人角蛋白的污染和人血浆蛋白的共存。

项目成果

Handling and optimization of urinary protein preparation for 2-DE based Proteomic analysis and 2D DIGE quantitative analysis of urine proteomes of healthy Male children and adults

用于基于 2-DE 的蛋白质组学分析和健康男性儿童和成人尿液蛋白质组的 2D DIGE 定量分析的尿蛋白制备的处理和优化

• 发表时间: 2007
• 作者: Gao SY;Li CY;Shimokawa T;Terashita T;Matsuda S;Yaoita E;Kobayashi N;Shono A;Miyamoto M;張榮;Zhang Y;Koda R;Zhao L;Fujinaka H;Zhao L;Miyamoto M;Yoshida Y
• 通讯作者: Yoshida Y

Role of Fat1 in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney

Fat1在嘌呤霉素氨基核苷肾病和新生儿肾足细胞接触形成中的作用

• 发表时间: 2005
• 期刊: Kidney Int 68
• 作者: Eishin;Yaoita
• 通讯作者: Yaoita

Two-Dimensional gel eiectrophoresis-besed proteome analysis of normal human Urine proteome: Optimization of urine sample handling and quantitative difference between healthy children and adults

正常人尿液蛋白质组的二维凝胶电泳蛋白质组分析：优化尿液样本处理以及健康儿童和成人之间的定量差异

• 发表时间: 2007
• 作者: Yutaka;Yosida
• 通讯作者: Yosida

Comprehensive preteome analysis of normal human kidney glomerulus with two dimensional pretein prefractionation in combination with nanoflow LC-MS/MS

通过二维蛋白预分离结合纳流 LC-MS/MS 对正常人肾小球进行全面的蛋白质组分析

• 发表时间: 2006
• 作者: Hirota M;Ishino K;Fukumasu I;Yoshida K;Mohri S;Shimizu J;Kajiya F;Sano S.;Miyamoto M;Miyazaki S;Nakahashi K;Hyodo T;Yoshikawa Y;Masahito Miyamoto
• 通讯作者: Masahito Miyamoto

Anti-perforin antibody treatment ameliorates experimental crescentic glomerulonephritis in WKY rats

抗穿孔素抗体治疗可改善 WKY 大鼠实验性新月体肾小球肾炎

• 发表时间: 2007
• 期刊: Kidney Int 72(2)
• 作者: Fujinaka H;Yamamoto T;Feng L;Nameta M;Garcia G;Chen S;El-shemi AA;Ohshiro K;Katsuyama K;Yoshida Y;Yaoita E;Wilson CB
• 通讯作者: Wilson CB