MODELS FOR EXPLORING FREE RADICAL DAMAGE

探索自由基损伤的模型

• 批准号: 2390069
• 负责人: Julie Kay Andersen
• 金额: $ 11.88万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-20 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390069
• 关键词: 6 hydroxydopamine; Parkinson's disease; aging; animal genetic material tag; brain metabolism; catalase; developmental neurobiology; enzyme activity; ferritin; genetically modified animals; glutathione peroxidase; human genetic material tag; hydrogen peroxide; iron; laboratory mouse; methylphenyltetrahydropyridine; neural degeneration; neuroanatomy; neuropharmacology; newborn animals; oxidative stress; substantia nigra; tissue /cell culture

Superoxide and hydrogen peroxide are produced during normal metabolism. However, when produced in excess, the oxidant species can be converted by reaction with free iron to reactive hydroxyl radicals which can degrade DNA, proteins, and membrane lipids resulting in cellular degeneration. The brain is particularly susceptible to free-radical damage due to its unique cellular membrane composition, and its high levels of oxygen metabolism and free iron. Brain cells have developed several defense against these oxidant species including production of antioxidant enzymes and storage of iron in forms that will not catalyze formation of reactive radicals. Specifically, the antioxidant enzyme superoxide dismutase reduces superoxide to hydrogen peroxide, which is in turn reduced by either glutathione peroxidase or catalase to H20. Iron, in turn, is normally bound to ferritin, the major iron storage molecule in the brain. However, when balance in the brain is disturbed and free-radicals are allowed to accumulate, this can lead to iron- catalyzed production of hydroxyl radical and subsequent cell damage. It has been postulated that oxidative stress may play a role in the pathogenesis of neurodegenerative diseases like Parkinson Disease (PD) and cellular degeneration during normal aging. To examine the role of antiodixants in preventing neuronal cell damage, transgenic mice will be create which over-express the iron-binding protein ferritin, or the anti- oxidant enzymes glutathione peroxidase or catalase in neurons using neuron-specific promoters. Constructs will be evaluated for their ability to confer transgene activity on cultured cells by Northern analysis, immunocytochemistry, enzyme assays, and for sensitivity to iron, MPTP, 6-OHDA, and H202. Following injection into mouse embryos, genomic insertion of constructs will be confirmed by PCR and Southern blot analysis. Transgenic mice will be bred to create new lines and progeny examined for transgene expression in brain by Northern analysis, in situ hybridization, immunocytochemistry, and enzyme assays. Transgenic lines will be evaluated during normal aging for changes in lipid peroxidation by MDA accumulation, protein oxidation by glutamine synthetase activity, oxidative stress as assessed by GSH/GSSG ratios, and for cell loss or atrophy as measured by cell number and sizes in the substantia nigra using tyrosine hydroxylase immunocytochemistry compared with normal controls. Levels of striatal dopamine and its metabolite, DOPAC, tyrosine hydroxylase activity, 3H-dopamine binding, and dopaminergic cell numbers and sizes will be assessed in transgenic compared to controls following MPTP injection. Changes in brain morphology during development, and in protecting against the effects of oxidative stress in the brain, exploring the hypothesis that a genetic increase in one of these molecules may be involved in predisposition to or protection against PD or neuronal degeneration during aging.

超氧化物和过氧化氢在正常代谢过程中产生。 然而，当过量产生时，氧化剂物质可被转化， 通过与游离铁反应生成反应性羟基自由基， 降解DNA、蛋白质和膜脂， 退化 大脑对自由基特别敏感 由于其独特的细胞膜组成， 氧代谢水平和游离铁。 脑细胞已经发育 针对这些氧化剂物质的几种防御措施包括产生 抗氧化酶和铁的储存形式， 反应性自由基的形成。 特别是抗氧化酶 超氧化物歧化酶将超氧化物还原为过氧化氢， 继而被谷胱甘肽过氧化物酶或过氧化氢酶还原成H2O。 反过来，铁通常与铁蛋白结合，铁蛋白是主要的铁储存器 大脑中的分子。 然而，当大脑的平衡被打破时， 自由基可以积累，这会导致铁- 催化羟基自由基的产生和随后的细胞损伤。 它 据推测，氧化应激可能在 帕金森病（PD）等神经退行性疾病的发病机制 和细胞退化。 审查的作用 在防止神经元细胞损伤的抗利尿剂，转基因小鼠将 产生过表达铁结合蛋白铁蛋白，或抗- 氧化酶谷胱甘肽过氧化物酶或过氧化氢酶在神经元中使用 神经元特异性启动子。 将评估构建体的 通过北方RNA转移酶对培养细胞赋予转基因活性的能力 分析，免疫细胞化学，酶测定，以及对 铁、MPTP、6-OHDA和H2 O2。 在注入小鼠胚胎后， 构建体的基因组插入将通过PCR和Southern杂交确认。 印迹分析 转基因小鼠将被培育成新的品系， 通过北方分析检测后代脑中的转基因表达， 原位杂交、免疫细胞化学和酶测定。 将在正常老化期间评价转基因品系的变化， 脂质过氧化（MDA积累）、蛋白质氧化（谷氨酰胺） 合成酶活性，通过GSH/GSSG比率评估的氧化应激，以及 对于细胞损失或萎缩，如通过细胞数量和大小测量的， 黑质酪氨酸羟化酶免疫细胞化学比较 与正常对照。 纹状体多巴胺及其代谢物的水平， DOPAC，酪氨酸羟化酶活性，3 H-多巴胺结合，和 多巴胺能细胞的数量和大小将在转基因小鼠中评估。 与MPTP注射后的对照相比。 大脑的变化 形态在发展过程中，并在保护免受影响， 大脑中的氧化应激，探索一种遗传的假设， 这些分子之一的增加可能涉及易感性， 或在衰老过程中防止PD或神经元变性。