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Transcriptional regulation of apoE DNA on dietary hyperlipidemia

apoE DNA 对饮食性高脂血症的转录调控

基本信息

批准号：
03671128
负责人：
MATSUSHIMA Teruhiko
金额：
$ 1.34万
依托单位：
The University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

In order to investigate the control mechanism of apolipoprotein E (apoE) biosynthesis, 5' regulatory region of guinea pig apoE genomic DNA was cloned in CAT vector, transfected into the primary cultured guinea pig hepatocytes and the transcriptional activity was analyzed by CAT assay system. DNA-nuclear protein interaction was analyzed by gel shift analysis, DNaseI footprint analysis and Southwestern blotting. On the CAT assay analysis, the apoE gene showed stronger transcriptional activity in hepatocytes from cholesterol-fed guinea pig than in the cells from normal animals. Gel shift analysis showed DNA-protein complex of the 5' upstream region of the gene with nuclear extract of the guinea pig liver. The binding patterns of the DNA were different between to the normal liver and to the fatty liver from animals fed with high cholesterol diet. In the latter the content of apoE mRNA had been confirmed to be increased, suggesting that the change of DNA-protein interaction may cause enhanced transcription. Southwestern blotting analysis was performed on the nuclear protein with 32P-labeled 5' upstream region of the DNA as a prove. Signals were detected at the 110 and 33 kilodaltons. The signal at 33kD was observed stronger in the extract from normal liver than in that from fatty liver. The 33kD protein may be involved in the positive transcriptional regulation of apoE gene at the cholesterol loading. It was suggested that apoE synthesis is up-regulated at the level of DNA transcription by cholesterol loading.

为了探讨载脂蛋白E （apoE）生物合成的调控机制，将豚鼠载脂蛋白E基因组DNA的5′调控区克隆到CAT载体上，转染到原代培养的豚鼠肝细胞中，利用CAT检测系统分析其转录活性。通过凝胶位移分析、dna印迹分析和西南印迹分析dna -核蛋白相互作用。在CAT分析中，apoE基因在喂食胆固醇的豚鼠肝细胞中的转录活性比在正常动物肝细胞中的转录活性强。凝胶位移分析显示该基因上游5'区dna -蛋白复合物与豚鼠肝脏核提取物。DNA的结合模式在正常肝脏和高胆固醇饮食动物的脂肪肝之间是不同的。后者证实apoE mRNA含量增加，提示dna -蛋白相互作用的改变可能导致转录增强。用32p标记的DNA上游5'区对核蛋白进行西南印迹分析作为证明。在110和33千道尔顿处检测到信号。正常肝提取物中33kD处的信号强于脂肪肝提取物。33kD蛋白可能参与apoE基因在胆固醇负荷时的正向转录调控。这表明载脂蛋白e的合成在DNA转录水平上受到胆固醇负荷的上调。