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

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基因 通过使用数量性状基因座 (QTL) 分析小鼠衰老。 为此，一些与氧化应激相关的分子标记 将在 150 岁的横断面研究中对整个生命周期进行测量， 450 和 750 天龄，使用 C57Bl/6 DBA/2 (BXD) 重组近交系 (RI)品系和BXD F2小鼠；这些标记的数量差异 然后将菌株和个体之间的差异与基因分型相关联 数据。本次重新提交对总体研究设计进行了重大修改 初步审查提出的宝贵建议扩大了范围 该子项目的分析方法。我们扩大了面板 氧化应激标记物，包括影响 3 个主要因素的标记物 包括大脑在内的 3 个器官中的大分子类别 研究表明，随着小鼠年龄的增长，其变化显着。具体来说， 心脏组织中 DNA 的氧化损伤将通过以下方式进行评估 通过 PCR 定量线粒体 DNA 缺失并测量 8 通过 HPLC 测定羟基-2'-脱氧鸟苷。抗氧化剂的蛋白质水平 含铜锌超氧化物歧化酶 (CuZn-SOD) 和 含有锰的超氧化物歧化酶（Mn-SOD）将在 通过蛋白质印迹检测肝脏。还将评估肝脏中的脂质过氧化 与硫代巴比妥酸测定。小脑中的蛋白质氧化 定量为蛋白质羰基含量。这些标记物的定量 氧化应激的程度将与多态性遗传相关 标记来识别影响这些年龄的染色体区域（QTL） 相关现象。