Remaley A T, Schumacher U K, Amouzadeh H R, Brewer H B, Hoeg J M
National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Lipid Res. 1995 Feb;36(2):308-14.
Several key genes involved in cholesterol metabolism are known to be directly regulated by cholesterol. The possible indirect effect, however, of increased levels of cellular cholesterol on gene expression and its possible role in cholesterol metabolism and atherosclerosis has not been thoroughly explored. In order to determine the overall effect of cholesterol on gene expression, we isolated differentially expressed genes from a PCR-based subtraction library prepared from the liver of chow-fed and cholesterol-fed rabbits. A total of nine upregulated and four down-regulated cDNA fragments were isolated. As determined by Northern blot analysis, the expression of the isolated cDNAs began to change as early as the first week on the cholesterol-rich diet or as late as 4 weeks, which corresponded with hepatic cholesterol accumulation. Three of the cDNAs were identified by DNA sequence homology, whereas the remaining cDNAs had no significant homology match. CYP1A1, a cytochrome P450 isoenzyme, was found to be down-regulated in hepatocytes by cholesterol feeding. Osteopontin and Mac-2, which are produced by macrophages, were found to be up-regulated in Kupffer cells by cholesterol feeding. Overall these results demonstrate the usefulness of the subtraction library approach for identifying new candidate genes for exploring the pathogenesis of atherosclerosis.
已知参与胆固醇代谢的几个关键基因直接受胆固醇调控。然而，细胞胆固醇水平升高对基因表达的可能间接影响及其在胆固醇代谢和动脉粥样硬化中的可能作用尚未得到充分探索。为了确定胆固醇对基因表达的总体影响，我们从以普通饲料喂养和胆固醇喂养的兔子肝脏制备的基于PCR的消减文库中分离出差异表达基因。总共分离出9个上调和4个下调的cDNA片段。通过Northern印迹分析确定，分离出的cDNA的表达最早在富含胆固醇饮食的第一周开始变化，最晚在4周时变化，这与肝脏胆固醇积累相对应。其中3个cDNA通过DNA序列同源性鉴定，而其余的cDNA没有显著的同源性匹配。细胞色素P450同工酶CYP1A1被发现通过胆固醇喂养在肝细胞中下调。巨噬细胞产生的骨桥蛋白和Mac-2被发现通过胆固醇喂养在库普弗细胞中上调。总体而言，这些结果证明了消减文库方法在鉴定用于探索动脉粥样硬化发病机制的新候选基因方面的有用性。
