Parkin表达上调对AD转基因果蝇模型神经保护作用及其机制的研究

发布时间：2018-06-27 07:55

本文选题：阿尔茨海默病 + Tau　；参考：《桂林医学院》2015年硕士论文

【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。
[Abstract]:Objective: Alzheimer's disease (AD) is a common neurodegenerative disease characterized by progressive cognitive impairment and memory impairment. The characteristic pathological changes are neurofibrillary tangles (NFT) formed by the deposition of 1 尾 -amyloid protein (A 尾) 2Tau protein hyperphosphorylation, which has become a hot spot in AD research in recent years. Parkin is a special protein. It is encoded by the PARK2 gene and has the activity of E3-ubiquitin protein ligase, which can link and mediate the ubiquification of the substrate protein. Therefore, the toxic protein can be degraded by Parkin and then from the ubiquitin proteasome pathway. At the same time, Parkin has the function of mediating mitochondrial division. Therefore, the damaged mitochondria are cleared through the mitochondrial autophagy pathway after mitochondrial division, so that the function of mitochondria can be improved. In this study, transgenic Drosophila melanogaster was used as AD model to investigate whether the up-regulation of Parkin expression had neuroprotective effect on AD and the mechanism of its neuroprotective effect. Methods: using the classical Gal4 / UAS system, ninaE-GAL4 promoter was used to selectively express Tau protein at R406W in Drosophila drosophila compound eye to construct AD transgenic Drosophila melanogaster model with ninaE-GAL4 / UAS system. The AD transgenic Drosophila model with up-regulation of Parkin expression and intervention of ninaE-GAL4 / UAS system was constructed by using Drosophila hybrida hybridization under the condition of knockout of mitochondrial cleavage protein Drp1 or not. Then the mRNA level of Parkinn Drp1 was detected, and the ATP content in brain tissue was detected by microscope and electron microscope. Results: AD transgenic Drosophila melanogaster model with ninaE-GAL4 / UAS system and AD transgenic Drosophila fly model with up-regulation of Parkin expression and intervention of ninaE-GAL4 / UAS system were successfully constructed. It was found that the drosophila melanogaster had irregular appearance, disappeared lines, decreased the area and the ATP content of brain tissue in AD transgenic model of ninaE-GAL4 / UAS system. However, the up-regulated Parkin gene was involved in the treatment of Drosophila melanogaster. It can be seen that the compound eye shape of Drosophila melanogaster is more regular than that of the former, and the ATP content in brain tissue is obviously higher than that of the model Drosophila melanogaster. However, after the interference of mitochondrial mitogen Drp1 RNA, the compound eye shape of AD transgenic model treated by Parkin is irregular and the lines are not clear. Compared with the former, the area decreased obviously, and the ATP content of brain tissue decreased obviously. ConclusionThe neuroprotective effect of 1: Parkin on AD transgenic Drosophila model may depend on the function of mitochondrial mitogen Drp1.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R749.16

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