【作者】 孟秀梅；

【导师】 王鲁宁；

【作者基本信息】 中国人民解放军军医进修学院 ， 老年神经病学， 2009， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是痴呆中最常见的一种,其发病机制及病理过程国内外学者通过人脑组织、动物模型、细胞模型等已经做了大量的研究,但其确切病因尚不明确。目前国内尚无AD相关的人脑蛋白质组学研究,本次实验利用中国人民解放军总医院老年医学研究所组织库收集的宝贵尸检病例标本进行研究。本次实验主要包括三个部分:1)利用HE、免疫组织化学方法对病例进行筛选,根据病理诊断标准确诊后,结合临床诊断分组;2)通过荧光双向差异凝胶电泳技术分离蛋白点,并利用质谱进行蛋白点鉴定,搜库并分析蛋白点在AD发病机制中的意义。3)选择部分蛋白点进行western blotting验证,证明双向电泳的稳定性和准确性。论文的第一部分是对AD的病理学筛查,利用国际公认的,发生特征性病理变化的抗体进行免疫组织化学染色,我们总共选择了anti-β-Amyloid/AT8/Synuclin/Ubiquitin四种抗体,根据淀粉样沉积、纤维缠结、神经毡等染色情况以及HE染色显示的神经元丢失等判断选择出AD病例,并利用Synuclin染色结果鉴别出同样具有痴呆表现的路易痴呆或帕金森病性痴呆。结合临床诊断标准最终选择出6例AD和6例正常对照,进行下一步的蛋白质组学研究。论文的第二部分主要是:根据第一部分结论分成两组的病例,提取对应部位的新鲜脑组织蛋白,并进行荧光双向差异凝胶电泳,将获得的6副三通道荧光图输入配套的图像分析软件Image Master 2D Platinum、Image Quant、DeCyder(Version6.5)进行分析,分析得到的差异蛋白点进行质谱鉴定,明确差异蛋白点性质,搜库分析其与AD的关系。在本次实验中共发现18个差异蛋白点,鉴定了其中17个,得到14个蛋白质,其中有两种蛋白质是由不同的蛋白点鉴定所得。经查阅大量文献,发现获得的蛋白质多为氧化应激相关、骨架蛋白、分子伴侣蛋白等,有4种蛋白质未在以往的研究中报道,而另外的蛋白质的含量变化也与国外研究报道的不尽相同。第三部分主要利用western blotting方法验证了热休克蛋白90α的含量变化,实验结果显示与双向电泳的结果一致,验证了双向电泳的稳定性和可靠性。本研究为以后的AD发病机制的研究,为AD的诊断及治疗提供了新思路。更多还原

【Abstract】 Alzheimer’s disease(AD) is one of the most nomal dementias.A lot of work to identify the pathogenetic and pathologic progress of AD have been done by investigators in many countries using brain tissues of people,animal models and cell models.Now in China there is no study about proteomic of AD brain of people.We carried the study in the Geronotlogical Research Center of the general hospital of PLA with the samples conserved in paraffin wax and in refrigerator at -80℃.The study included three parts:1)we selected the samples of AD and samples of control by HE stain and immunohistochemistry stain.Combining the clinical diagnosis,we designed the two groups after we obtained the pathologic diagnosis; 2) we isolated proteins of the two groups(AD group and control group) by fluorescence two-dimensional differential gel electrophoresis(2D-DIGE).Then identified the proteins by mass spectrometry and analyzed the association between the AD pathogenesy and the proteins.3)at the end,we validated part of the altered proteins by western blotting.The first part:pathobiology.Anti-β-Amyloid,anti-AT8,anti-Ubiquitin were used to obtain the AD samples and contol samples,which are the most common antibodies admitted by people to characterize the AD’s pathological changes.We used anti-Synuclin to exclude Lewy’ body dementia and Parkinson’s dementia. With all these pathological changes we at length defined two groups:AD group and control group with each group including 6 cases.The second part:proteom study.After extracted protein from the cases of two groups,we ran the 2D-DIGE.The protein spots were analyzed by softwares of Image Master 2D Platinum,Image Quant and DeCyder(Version6.5) and then were identified by mass spectrometry.After searching in IPI HUMAN(v3.23) database,we obtained 14 different proteins.The different proteins are of oxidation, energy-related enzymes,and chaperonin et al.4 of the proteins have not been reported before in the studies of other investigators.Some alteration of protein level didn’t correspond with that of the previous studies. The third part of the study was to validate the results of 2D-DIGE with western blotting.We detected the altered level of HSP90αand the two results corresponded.This demonstrated the stability and reliability of the 2D-DIGE.This study leads a new road to ascertain the pathogenesy of AD,and to find new diagnosis and therapy methods to AD.更多还原