QUANTITATIVE TRAIT LOCI ANALYSIS OF OXIDATIVE STRESS MARKERS

氧化应激标记物的定量性状位点分析

• 批准号: 6299397
• 负责人: Glenn S Gerhard
• 金额: $ 17.67万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6299397
• 关键词: DNA damage; aging; animal genetic material tag; cerebellum; deoxyguanosine; genetic markers; genetic polymorphism; genotype; high performance liquid chromatography; laboratory mouse; linkage mapping; lipid peroxides; liver; mitochondrial DNA; myocardium; oxidative stress; polymerase chain reaction; quantitative trait loci; superoxide dismutase; western blottings; DNA damage; aging; animal genetic material tag; cerebellum; deoxyguanosine; genetic markers; genetic polymorphism; genotype; high performance liquid chromatography; laboratory mouse; linkage mapping; lipid peroxides; liver; mitochondrial DNA; myocardium; oxidative stress; polymerase chain reaction; quantitative trait loci; superoxide dismutase; western blottings

The long-term goal of this proposal is to identify genes influencing murine aging through the use of quantitative trait loci (QTL) analysis. To do so, several molecular markers associated with oxidative stress will be measured across the lifespan in a cross-sectional study at 150, 450, and 750 days of age using C57Bl/6 DBA/2 (BXD) recombinant inbred (RI) strains and BXD F2 mice; quantitative differences in these markers among strains and individuals will then be correlated with genotyping data. In this resubmission, major revisions in the overall study design and valuable suggestions by the initial reviews have led to a broadening of the analytic approach for this subproject. We have expanded the panel of oxidative stress markers to include those which affect 3 major classes of macromolecules in 3 organs, including brain, and which have been shown to change significantly with age in mice. Specifically, oxidative damage to DNA in cardiac tissue will be assessed by quantitation of mitochondrial DNA deletions by PCR and measurement of 8 hydroxyl-2'-deoxyguanosine by HPLC. Protein levels of the antioxidant enzymes copper-zinc containing superoxide dismutase (CuZn-SOD) and manganese containing superoxide dismutase (Mn-SOD) will be measured in liver by Western blot. Lipid peroxidation will also be assessed in liver with the thiobarbituric acid assay. Protein oxidation in cerebellum will be quantified as protein carbonyl content. Quantitation of these markers of oxidative stress will then be correlated with polymorphic genetic markers to identify chromosomal regions (QTL) influencing these age- related phenomena.

该提案的长期目标是确定影响的基因 通过使用定量性状基因座（QTL）分析，鼠老化。 为此，与氧化应激相关的几个分子标记 将在150的横断面研究中在整个寿命中测量， 使用C57BL/6 DBA/2（BXD）重组近近交易的450天和750天的年龄 （RI）菌株和BXD F2小鼠；这些标记的定量差异 在菌株和个体中，将与基因分型相关 数据。在此重新提交中，整体研究设计中的重大修订 初步评论的宝贵建议导致了扩大 该副本的分析方法。我们扩展了面板 氧化应激标记的包括影响3大的氧化应力标记 包括大脑在内的3个器官中的大分子类 显示出随着小鼠的年龄而发生的显着变化。具体来说， 心脏组织中DNA的氧化损伤将通过 通过PCR定量线粒体DNA缺失，测量8 HPLC的羟基-2'-脱氧鸟苷。抗氧化剂的蛋白质水平 含有超氧化物歧化酶（Cuzn-SOD）和 将测量含有锰的超氧化物歧化酶（MN-SOD）的 Western印迹的肝脏。脂质过氧化也将在肝脏中进行评估 带有硫巴比妥酸测定法。小脑中的蛋白质氧化会 被量化为蛋白质羰基含量。这些标记的定量 然后，氧化应激将与多态性遗传相关 标记以鉴定影响这些年龄的染色体区域（QTL） 相关现象。