衰老及相关疾病中线粒体损伤与保护机制研究

发布时间：2017-12-28 10:46

本文关键词：衰老及相关疾病中线粒体损伤与保护机制研究　出处：《第二军医大学》2006年博士论文　论文类型：学位论文

【摘要】：在阐述衰老现象的多种理论中,自由基衰老理论近年来受到广泛重视,得到众多实验证据支持。自由基衰老理论认为,在衰老及相关疾病(如帕金森病(Parkinson's disease,PD),阿尔茨海默病(Alzheimer's disease,AD))中,自由基对细胞内生物大分子(核酸、脂类、蛋白等)产生氧化损伤,损伤随年龄累积从而损害细胞功能,导致细胞功能衰退或丧失,最终造成机体衰老及老年性疾病。自由基中最重要的一类活性分子是含氧自由基,即活性氧(reactive oxygen species,ROS),线粒体(特别是损伤线粒体)是细胞内产生ROS的主要细胞器,同时线粒体自身富含的多种酶、结构蛋白、膜脂质及核酸等也是ROS直接攻击的目标,受损线粒体ROS产生增加,造成线粒体内氧化损伤的恶性循环。对于衰老及相关疾病中,如何简便定量线粒体基因中的异质性/点突变:在ROS(如活性较强的羟自由基,·OH)作用下线粒体呼吸功能及线粒体中相关抗氧化系统如何变化;ROS引发线粒体内膜脂质过氧化后,脂质过氧化产物(MDA)累积是否进一步损伤线粒体呼吸链及氧化磷酸化相关酶;依据线粒体营养素假说,在衰老及相关疾病(PD)所致的线粒体功能损伤中,如何靶向性干预和纠正线粒体氧化损伤;线粒体营养素(如硫辛酸)在衰老及PD动物模型中,如何缓解相关症状,其可能的生化机制如何。以上内容还缺乏足够的实验研究。针对以上问题,本研究以线粒体为研究重点,从线粒体核酸、酶学、特征蛋白表达等方面在体外及活体模型作了以下几方面探索: 一.改良PCR-RFLP法测定异质性线粒体基因相对含量发生线粒体病变的细胞或受到活性氧攻击的线粒体中往往存在线粒体基因异质性现象,测定异质性线粒体基因的相对含量对于了解线粒体基因功能及基因诊断具有一定的意义。我们在研究ECV304细胞线粒体基因D-LOOP区过程中,通过克隆测序,发现在ECV304细胞线粒体基因513碱基存在A/G异质性,用传统PCR-RFLP、3′端特异引物法无法测定异质性基因的相对含量。通过对传统PCR-RFLP法的改进,依据ECV304细胞中线粒体基因(D-LOOP区)异质性位点,采用两轮PCR法,第一轮PCR胶回收产物为模板,设计半巢式引物作第二轮PCR,并在第二轮PCR中引入限制性内切酶位点,酶切产物行琼脂糖电泳,测定异质性线粒体基因的相对含量。发现ECV304细胞中存在513A/G异质性,二者比例约为1/3。改良PCR-RFLP法测定异质性线粒体基因相对含量较为简单可靠,适用于异质性线粒体基因的研究。
[Abstract]:In the various theories of aging, the theory of free radical senescence has been paid much attention in recent years and has been supported by many experimental evidence. That the free radical theory of aging, the aging and related diseases (such as Parkinson's disease (Parkinson's disease PD), Alzheimer's disease (Alzheimer's, disease, AD)), free radicals on intracellular biological macromolecules (proteins, nucleic acids, lipids etc.) cause oxidative damage, damage and damage accumulation with age leads to cell function. Decline or loss of cell function, resulting in aging and age-related diseases. A class of active molecules, the most important is the free radicals of oxygen radicals, namely, reactive oxygen species (reactive oxygen species, ROS), mitochondria (especially damaged mitochondria) is the main organelle producing ROS, and mitochondrial enzymes, their rich structural proteins, membrane lipid and nucleic acid is ROS direct attack the target damaged mitochondrial ROS production increased, resulting in oxidative damage to mitochondria in the vicious spiral. For aging and related diseases, how simple heterogeneity / quantitative point mutations in the mitochondrial gene in ROS (such as the strong activity of hydroxyl radicals, OH) how relevant antioxidant system and mitochondrial respiratory function under the effect of changes in mitochondrial membrane lipid; ROS induced peroxidation, lipid peroxidation (MDA) whether the accumulation of further damage to the mitochondrial respiratory chain and oxidative phosphorylation enzymes; based on the hypothesis of mitochondrial nutrients in aging and related diseases (PD) caused by the damage of mitochondrial function, how to targeted intervention and correction of mitochondrial oxidative damage; mitochondrial nutrients (such as lipoic acid) in aging and PD animal model, how to how to relieve the symptoms, the possible biochemical mechanisms. The above content is still lack of sufficient experimental research.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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