QUANTITATIVE TRAIT LOCI ANALYSIS OF OXIDATIVE STRESS MARKERS

这项提议的长期目标是确定影响 通过利用数量性状基因座(QTL)分析小鼠的衰老。 要做到这一点，几个与氧化应激有关的分子标记 将在150岁时的横断面研究中测量寿命， C57BL/6 DBA/2(BXD)重组自交系450、750日龄 (RI)品系和BXD F2小鼠；这些标记的数量差异 然后在菌株和个体之间进行基因分型 数据。在这次重新提交中，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导致了范围的扩大 这一子项目的分析方法。我们扩大了小组讨论范围 氧化应激标志物包括那些影响3个主要 包括大脑在内的3个器官中的大分子类别，它们有 已被证明在小鼠身上随着年龄的增长而显著变化。具体来说， 对心脏组织DNA的氧化损伤将通过以下方法进行评估 用聚合酶链式反应检测线粒体DNA缺失及8 高效液相色谱法测定羟基-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 含锰超氧化物歧化酶(Mn-SOD)将在 用免疫印迹法检测肝脏。肝脏中的脂质过氧化也将被评估。 用硫代巴比妥酸测定。小脑中的蛋白质氧化将 被量化为蛋白质的羰基含量。这些标记物的定量 氧化应激的程度将与多态遗传相关 识别影响这些年龄的染色体区域(QTL)的标记- 相关现象。

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.