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Analysis of aging-associated genes in myocardium : application of RT-PCR differential display method.

心肌衰老相关基因分析：RT-PCR差异显示法的应用。

基本信息

批准号：
09557008
负责人：
KATANO Yumi
金额：
$ 7.49万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

It has been known that the physiological function of the heart is altered according to the aging. For example, the positive inotropic effects of isoproterenol and endothelin-1 are decreased by aging in the rat ventricles. While the binding capacity for b-adrenergic receptor was decreased, the specific binding capacity of endothelin-1 was increased in the aged rats. In sarcoplasmic reticulum, the activity of CaィイD12+ィエD1 was reduced. In coronary arterial endothelium, nitric oxide production was very low-in aged rat. The molecular basis of these age-dependent functional changes has not been understood. Differential gene expression provides the basis for many fundamental cellular processes associated with differentiation, development and aging. The identification and cloning of genes whose expression is modulated is an important step in advancing our understanding of aging phenomena. In order to isolate and characterize gene products differentially expressed in aging, we applied the diffe … More rential display reverse transcription-polymerase chain reaction for screening those genes in 2-3 months-old and 27 months-old Fischer 344 rat ventricles. Total RNAs were isolated by acid-guanidium-phenol-chloroform method. Messenger RNAs were obtained from total RNA by using oligo d(T) column. The first-strand cDNAs from each RNA were synthesized with three oligo-d(T)-anchor primers. PCR was carried out with anchor primers and eight arbitrary primers, and the products were analyzed on sequence gels. The 19 unique PCR products displayed increased expression in aged heart, while 66 clones were decreased in aged heart. Differential expression was confirmed by Northern blot analysis or reverse Northern blotting. The clones included genes for mitochondrial metabolism such as cytochrome c oxidase, myosin heavy chain, cell surface glycoproteins and several kinases/phosphatases for signal transduction. 31 clones were sequenced and 14 clones were unknown genes. The method used in this study is valuable for studying the age-associated gene expression, and we are continuing to analyze the unknown genes identified in this study. Less

众所周知，心脏的生理功能随着年龄的增长而改变。例如，异丙肾上腺素和内皮素-1的正性肌力作用在大鼠心室中随着年龄的增长而降低。老年大鼠内皮素-1的特异性结合量增加，而β-肾上腺素能受体结合量减少。肌浆网中Ca ~（2+）D_12 + Ca ~（2+）D_1活性降低。在老年大鼠冠状动脉内皮细胞中，一氧化氮的产生非常低。这些年龄依赖性功能变化的分子基础尚未被理解。差异基因表达为许多与分化、发育和衰老相关的基本细胞过程提供了基础。识别和克隆基因的表达是调节是一个重要的一步，在推进我们对衰老现象的理解。为了分离和鉴定衰老过程中差异表达的基因产物，我们应用了差异表达的基因产物， ...更多信息 RT-PCR技术在2-3月龄和27月龄Fischer 344大鼠心室中筛选这些基因。采用酸-胍-酚-氯仿法提取总RNA。利用oligo d（T）柱从总RNA中提取信使RNA。用三个oligo-d（T）-锚引物合成来自每个RNA的第一链cDNA。用锚引物和8个随机引物进行PCR扩增，产物在序列凝胶上进行分析。19个特异性PCR产物在老年心脏中表达增加，而66个克隆在老年心脏中表达减少。通过北方印迹分析或反向北方印迹证实差异表达。这些克隆包括线粒体代谢基因，如细胞色素c氧化酶、肌球蛋白重链、细胞表面糖蛋白和几种信号转导激酶/磷酸酶。对31个克隆进行测序，其中14个克隆为未知基因。本研究中使用的方法对于研究年龄相关基因的表达是有价值的，我们正在继续分析本研究中发现的未知基因。少