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IDENTIFICATION OF GENES REGULATED BY ADVANCED GLYCATION END PRODUCTS IN CULTURED GLOMERULAR MESANGIAL CELLS

培养的肾小球系膜细胞中高级糖化终产物调控的基因的鉴定

基本信息

批准号：
08671147
负责人：
HANEDA Masakazu
金额：
$ 1.47万
依托单位：
SHIGA UNIVERSITY OF MEDICAL SCIENCE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Nonenzymatic glycation is proposed to be one of the etiologic factors of diabetic nephropathy. Although receptors for advanced glycation end products (AGEs) were found in glomerular mesangial cells, AGEs-induced changes in cellular functions have not been clarified yet. We performed mRNA differential display analysis, to identify AGEs-induced alterations of gene expression in cultured rat mesangial cells. Confluent mesangial cells were incubated with AGE-BSA (200mug/ml) or control BSA (200mug/ml) for 48 hours and total RNA was extracted. After reverse transcription with T12MN primers, cDNA was amplified by polymerase chain reaction using T12MN primers and random 10 mer, and the products were analyzed on a sequence gel. Of 6,000 mRNAs screened, 78 candidate PCR products were detected on a sequence gel. Northen blot analysis confirmed 6 clones changed by AGEs. Three of them were increased by AGEs and three clones were decreased. Two of three clones increased by AGEs showed homology to mitochondrial elongation factor Tu and mesenchyme fork head-1, respectively. One of three clones decreased by AGEs showed homology to actin depolymerizing factor. These three genes have been reported to be involved in protein synthesis or cell proliferation. Further analysis of these genes may provide new insight on pathogenesis of diabetic nephropathy. In conclusion, three genes regulated by AGEs in mesangial cells were identified by mRNA differential display analysis.

非酶糖基化被认为是糖尿病肾病的病因之一。尽管在肾小球系膜细胞中发现了晚期糖基化终产物（AGEs）受体，但AGEs诱导的细胞功能变化尚未阐明。我们进行了mRNA差异显示分析，以确定AGEs诱导的基因表达的改变，在培养的大鼠系膜细胞。将汇合的系膜细胞与AGE-BSA（200 μ g/ml）或对照BSA（200 μ g/ml）孵育48小时，并提取总RNA。在用T12 MN引物逆转录后，使用T12 MN引物和随机10 mer通过聚合酶链反应扩增cDNA，并在序列凝胶上分析产物。在筛选的6，000个mRNA中，在序列凝胶上检测到78个候选PCR产物。Northen blot分析证实有6个克隆被AGEs改变。AGEs使其中3个克隆表达增加，3个克隆表达减少。AGEs增加的三个克隆中有两个分别与线粒体延伸因子Tu和间充质叉头-1同源。AGEs减少的3个克隆中有1个克隆与肌动蛋白解聚因子同源。据报道，这三个基因参与蛋白质合成或细胞增殖。对这些基因的进一步分析可能为糖尿病肾病的发病机制提供新的见解。总之，通过mRNA差异显示分析，在系膜细胞中发现了三个受AGEs调控的基因。