The Carbonyl stress as an Etiology of Diabetic Microangiopathy. -Gene-targeting of the Enzyme Metabolizing Glucose-derived carbonyl compounds-

羰基应激作为糖尿病微血管病的病因。

• 批准号: 13671193
• 负责人: MIYATA Satoshi
• 金额: $ 2.11万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671193/
• 关键词: diabetes complications; glycation; methylglyoxal; glyoxalase-1; dicarbonyl compound; gene targeting

It has been known that the formation of highly reactive dicaibonyl compounds such as metliylglyoxal (MG), an intermediate of the gbycation reaction, is accelerated in diabetic subjects. Several lines of evidence have shown that excess MG influences cell fimctions in vitro, suggesting its involvement in the development of diabetic microangiopatny. On the other hand, it has been proposed that there is a defense system against MG by metabolizing it to an inert agent in vivo. In this regard, previous studies have shown that the glyoxalase system comprising glyoxalase-1 (GLO1) and gtyoxalase-2 plays a major role in it Thus, the present study was designed to clarify the relationship between MG metabolism and alteration in miciovascular tissues by establishing GLO1-knockout mice. In 2001, we first prepared a probe to clone mouse GLO1 cDNAby conducting PCR on mouse liver cDNA library with a set of primer deduced from the known human GLO1 cDNA sequence. Using this probe, we successfully cloned a full-length of mouse GL01 cDNA with an open reading frame of 552 bp whose sequence was 82% identical to that of human. The deduced amino acid sequence exhibits 89% identity with that of human. In 2002, we also found a silent SNP (225T/C) in the cloned GLO1 cDNAs isolated from mouse liver cDNA library. We subsequently screened a genome GLO1 DNA from a mouse genome DNA library with a probe including the start codon. Analysis of its sequence is now in progress. We are going to constmct a targeting vector aiming homologous replacement, followed by establishing GLO1 knockout mice. Finally, the mice will be analyzed in terms of the relationship between MG metabolism and alterations occurring in the imcrovasculatuie tissues.

众所周知，高活性二羰基化合物的形成，如甲基乙二醛（MG），一种甲基化反应的中间产物，在糖尿病患者中会加速。一些证据表明，过量的MG在体外影响细胞功能，提示其参与糖尿病微血管病变的发展。另一方面，有人提出存在一种防御系统，通过在体内将MG代谢为惰性剂来抵抗MG。在这方面，以往的研究表明，由glyoxalase-1 （GLO1）和gtyoxalase-2组成的glyoxalase系统在其中起主要作用，因此，本研究旨在通过建立GLO1敲除小鼠来阐明MG代谢与微血管组织改变之间的关系。2001年，我们首先制备了一种探针，利用已知人类GLO1 cDNA序列推导出的引物对小鼠肝脏cDNA文库进行PCR克隆小鼠GLO1 cDNA。利用该探针，我们成功克隆出了全长552 bp的小鼠GL01 cDNA，其序列与人类的同源性为82%。推导出的氨基酸序列与人类的同源性达到89%。2002年，我们还从小鼠肝脏cDNA文库中分离克隆的GLO1 cDNA中发现了一个沉默SNP （225T/C）。随后，我们用包含起始密码子的探针从小鼠基因组DNA文库中筛选了基因组GLO1 DNA。对其序列的分析正在进行中。我们将构建同源置换的靶向载体，然后建立GLO1敲除小鼠。最后，对小鼠进行MG代谢与血管组织变化之间的关系分析。