本文關(guān)鍵詞：衰老及相關(guān)疾病中線粒體損傷與保護(hù)機(jī)制研究　出處：《第二軍醫(yī)大學(xué)》2006年博士論文　論文類型：學(xué)位論文

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【摘要】：在闡述衰老現(xiàn)象的多種理論中,自由基衰老理論近年來受到廣泛重視,得到眾多實(shí)驗(yàn)證據(jù)支持。自由基衰老理論認(rèn)為,在衰老及相關(guān)疾病(如帕金森病(Parkinson's disease,PD),阿爾茨海默病(Alzheimer's disease,AD))中,自由基對細(xì)胞內(nèi)生物大分子(核酸、脂類、蛋白等)產(chǎn)生氧化損傷,損傷隨年齡累積從而損害細(xì)胞功能,導(dǎo)致細(xì)胞功能衰退或喪失,最終造成機(jī)體衰老及老年性疾病。自由基中最重要的一類活性分子是含氧自由基,即活性氧(reactive oxygen species,ROS),線粒體(特別是損傷線粒體)是細(xì)胞內(nèi)產(chǎn)生ROS的主要細(xì)胞器,同時(shí)線粒體自身富含的多種酶、結(jié)構(gòu)蛋白、膜脂質(zhì)及核酸等也是ROS直接攻擊的目標(biāo),受損線粒體ROS產(chǎn)生增加,造成線粒體內(nèi)氧化損傷的惡性循環(huán)。對于衰老及相關(guān)疾病中,如何簡便定量線粒體基因中的異質(zhì)性/點(diǎn)突變:在ROS(如活性較強(qiáng)的羥自由基,·OH)作用下線粒體呼吸功能及線粒體中相關(guān)抗氧化系統(tǒng)如何變化;ROS引發(fā)線粒體內(nèi)膜脂質(zhì)過氧化后,脂質(zhì)過氧化產(chǎn)物(MDA)累積是否進(jìn)一步損傷線粒體呼吸鏈及氧化磷酸化相關(guān)酶;依據(jù)線粒體營養(yǎng)素假說,在衰老及相關(guān)疾病(PD)所致的線粒體功能損傷中,如何靶向性干預(yù)和糾正線粒體氧化損傷;線粒體營養(yǎng)素(如硫辛酸)在衰老及PD動(dòng)物模型中,如何緩解相關(guān)癥狀,其可能的生化機(jī)制如何。以上內(nèi)容還缺乏足夠的實(shí)驗(yàn)研究。 針對以上問題,本研究以線粒體為研究重點(diǎn),從線粒體核酸、酶學(xué)、特征蛋白表達(dá)等方面在體外及活體模型作了以下幾方面探索: 一.改良PCR-RFLP法測定異質(zhì)性線粒體基因相對含量 發(fā)生線粒體病變的細(xì)胞或受到活性氧攻擊的線粒體中往往存在線粒體基因異質(zhì)性現(xiàn)象,測定異質(zhì)性線粒體基因的相對含量對于了解線粒體基因功能及基因診斷具有一定的意義。我們在研究ECV304細(xì)胞線粒體基因D-LOOP區(qū)過程中,通過克隆測序,發(fā)現(xiàn)在ECV304細(xì)胞線粒體基因513堿基存在A/G異質(zhì)性,用傳統(tǒng)PCR-RFLP、3′端特異引物法無法測定異質(zhì)性基因的相對含量。通過對傳統(tǒng)PCR-RFLP法的改進(jìn),依據(jù)ECV304細(xì)胞中線粒體基因(D-LOOP區(qū))異質(zhì)性位點(diǎn),采用兩輪PCR法,第一輪PCR膠回收產(chǎn)物為模板,設(shè)計(jì)半巢式引物作第二輪PCR,并在第二輪PCR中引入限制性內(nèi)切酶位點(diǎn),酶切產(chǎn)物行瓊脂糖電泳,測定異質(zhì)性線粒體基因的相對含量。發(fā)現(xiàn)ECV304細(xì)胞中存在513A/G異質(zhì)性,二者比例約為1/3。改良PCR-RFLP法測定異質(zhì)性線粒體基因相對含量較為簡單可靠,適用于異質(zhì)性線粒體基因的研究。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R363

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 張春梅;檳榔花提取物抗衰老作用研究[D];湖南農(nóng)業(yè)大學(xué);2011年

2 李曉瑞;用于細(xì)胞內(nèi)自由基區(qū)域定位識別的新型熒光探針的設(shè)計(jì)、合成及應(yīng)用[D];山東師范大學(xué);2011年

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本文編號：1345644