【作者】 黄强；

【导师】 李清华；

【作者基本信息】 桂林医学院 ， 神经病学， 2015， 硕士

【摘要】 目的:阿尔茨海默病(Alzhemier,s disease,AD)是一种常见的神经系统退行性疾病,临床主要特征为进行性认知功能障碍及记忆力减退。其特征性病理学改变为:①β-淀粉样蛋白的沉积(Aβ)②Tau蛋白过度磷酸化所形成的神经纤维缠结(NFT),而Tau蛋白过度磷酸化已成为近年来研究AD的一个热点。Parkin是一种特殊的蛋白,它由PARK2基因所编码,该种蛋白具有E3泛素-蛋白连接酶活性,可连接并介导底物蛋白泛素化,因此,毒性蛋白可以通过Parkin进而从泛素蛋白酶体通路降解。与此同时,Parkin具有介导线粒体分裂的功能,因此,受损的线粒体分裂后,再通过线粒体自噬途径被清除,从而达到提高线粒体功能的作用。本实验选用转基因果蝇作为AD模型来探讨表达上调Parkin是否对AD具有神经保护作用,以及研究其发挥神经保护作用的机制。方法:利用经典的GAL4/UAS系统,选用ninaE-GAL4启动子,将突变位点在R406W处的Tau蛋白在果蝇复眼内选择性表达,构建ninaE-GAL4/UAS系统AD转基因果蝇模型。分别在敲除线粒体分裂蛋白Drp1与否的情况下,通过果蝇杂交技术,构建Parkin表达上调干预ninaE-GAL4/UAS系统的AD转基因果蝇模型。然后分别进行Parkin、Drp1的mRNA水平含量的检测,果蝇复眼光镜、电镜下观察,检测脑组织ATP含量。结果:成功构建ninaE-GAL4/UAS系统的AD转基因果蝇模型及Parkin表达上调干预ninaE-GAL4/UAS系统的AD转基因果蝇模型。发现ninaE-GAL4/UAS系统AD转基因模型果蝇复眼外形不规则,纹路消失,面积较前者明显减小、脑组织ATP含量明显减少,而表达上调的Parkin基因干预疾病模型果蝇后,可见果蝇的复眼外形较前规则、脑组织ATP含量较模型果蝇明显增加,而在线粒体分裂蛋白Drp1 RNA干扰后,Parkin干预的AD转基因模型果蝇复眼外形较前不规则,纹路不清晰,面积较前者明显减小、脑组织ATP含量明显减少。结论:Parkin对AD转基因果蝇模型具有神经保护作用,而这种神经保护作用可能需要依赖线粒体分裂蛋白Drp1的功能。更多还原

【Abstract】 Objective:Alzheimer’s disease(AD),a common neurodegenerative disease, is characterized clinically by a progressive loss of cognition impairment and memory function.The characteristics of pathological changes including: ① β-amyloid deposition ②The hyperphosphorylation of Tau protein lead to neurofibrillary tangles(NFT).The excessive phosphorylated of Tau protein, However, has become a hot topic about the AD in recent years. Parkin,a particular protein, encoding by PARK2, is an ubiquitin protein ligase of E3.Parkin mediate Ubiquitination of substrate proteins,so that toxic protein, mediating Ubiquitination by Parkin, was degraded; what’s more, Parkin, mediating mitochondrial fission, contributes to the clearance of impaired mitochondria by mitochondrial autophagy pathway to improve mitochondrial function. In this experiment,we, using a AD transgenic Drosophila model, investigate whether upregulated expression of Parkin can play a neuroprotective role in AD and how it works. Methods:Here,using the classical model of GAL4/UAS system,and ninaE- GAL4/UAS system AD transgenic Drosophila models were constructed by using the promoter ninaE-GAL4,which drive hTauR406 W protein( Tau protein which mutation site is at R406W),selectively expression in cells of Drosophila eyes.Then,Parkin protein was expressed in AD transgenic Drosophila models by genetic methods with or without in a background of mitochondrial fission protein of Drp1.Then we detect the mRNA level of Drp1 and Parkin,using the real time-PCR.The change of drosophila compound eyes were also observed by us, using the light microscope and electron microscope.we also detect the ATP level of the head of drosophila. Results:AD transgenic drosophila model of the ninaE-GAL4/UAS system was constructed by us successfully, and then we expressed upregulation of Parkin in this model successfully. We found that the significant retinal structure was destroyed,ATP content of the brain of AD transgenic drosophila model of the ninaE-GAL4/UAS system was decreased. However, after upregulating the expression of Parkin in this model, the significant retinal structure was restored, the ATP content of the brain was improved. however,with the ATP content of the brain decreased,the significant retinal structure was destroyed again in a background of knockdown of mitochondrial fission protein of Drp1.Con clusion:It is confirmed that expression of Parkin could protect AD transgenic Drosophila models,and the protection role of Parkin is Drp1- dependent,maybe.更多还原