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同济大学 硕士学位论文 摘要
摘要
阿尔茨海默病（Alzheimer’s disease, AD），又称老年痴呆症，是一种常见的神经退行性疾病，表现出中枢神经系统淀粉样斑块的沉积和记忆缺失。多种AD模型小鼠被用于模拟及研究AD的发病过程和开发治疗AD的药物，其中以APP(swe)/PSEN1dE9迄今最为常用。近期报道Tg6799 AD小鼠模型病理改变发生更早，而该小鼠模型是否具有APP(swe)/PSEN1dE9小鼠类似的病理改变及认知异常，两种小鼠模型的病程发展有何不同，尚无相关报道。我们系统性比较了两种小鼠在特定月龄下AD病变特点：行为学异常、脑组织病理及生化指标改变及血清中Aβ水平。结果发现1) 4月龄Tg6799小鼠水迷宫潜伏期比野生型(Wild Type WT)显著缩短（26.9±5.0s vs 34.8±3.2s，训练第4天），而同月龄APP(swe)/PSEN1dE9小鼠与其WT相比无明显差异。2) 4月龄Tg6799小鼠脑中Aβ数量和面积比APP(swe)/PSEN1dE9小鼠明显增加：额叶皮层分别为16.2和61.4倍；海马为8.0和26.4倍。相比APP(swe)/PSEN1dE9小鼠，Tg6799小鼠在下丘脑有大量Aβ沉淀。3) 4、6、9月龄APP(swe)/PSEN1dE9小鼠血清中Aβ含量分别为3.5、3.1、4.7 ng/ml，在Tg6799小鼠在血清中未能检测到Aβ。结果表明，Tg6799小鼠发病早，病程进展快，病理改变严重，使用该小鼠进行AD研究可以大大缩短研究周期；两种小鼠模型在血清中Aβ含量及脑组织中的淀粉样斑块分布区域存在不同。以上特点为选择恰当的AD动物实验模型及检测指标提供了参考。
关键词：阿尔茨海默病，老年痴呆症，Tg6799，APP，PSEN1，APP(swe)/PSEN1dE9，行为学，Thioflavin S染色，Aβ ELISA
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Tongji University Master of Philosophy Abstract
ABSTRACT
Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by memory loss and amyloid ?????? protein deposition in CNS. In order to study AD pathology and develop AD therapeutic drugs, many AD mouse model have been established. Currently, APP (swe)/PSEN1dE9 is the most commonly used AD mouse model. Tg6799 mouse was recently reported to develop AD pathology at earlier age. However, whether Tg6799 mice have similar pathological and cognitive impairments as APP(swe)/PSEN1dE9 was unclear. We did the systematic comparison of behavior defect, pathological change in the brain and the Aβ level within serum in both Tg6799 and APP(swe)/PSEN1dE9 mice at their different age (4, 6, 9-month old). The results showed that: 1） In Morris water maze test, the latency of 4-month old Tg6799 is longer than its WT control (26.9±5.0s vs 34.8±3.2s at Day4), however, no difference was observed when comparing 4 month old APP(swe)/PSEN1dE9 with its WT littermates. 2）At 4-month old, Tg6799 mice develop much more Aβ plaque in the brain compared to APP (swe)/PSEN1dE9 (both plaque number and area), the ratio between two mice are: 16.2 (number) and 61.4 (area) folds in cortex, 8.0 (number) and 26.4 (area) folds in hippocampus. Besides, Tg6799 mice showed many Aβ deposition in thalamus. 3) The serum Aβ level of APP (swe)/PSEN1dE9 is 3.5 ng/ml, 3.1 ng/ml, 4.7 ng/ml at 4、6、9-month old respectively, which is not detected in Tg6799. The results suggest that the pathological feature of Tg6799 appears much earlier and more severe than APP(swe)/PSEN1dE9. Using Tg6799 as a mouse model for AD research can shorten the duration of investigation; meanwhile our results also indicate that the serum Aβ level and Aβ deposition area in brain are different between two mouse models. These data offer important references for selecting appropriate AD mouse model and corresponding pathological factors for AD research.
Key words: Alzheimer’s disease, Tg6799, APP, PSEN1, /PSEN1
dE9
, behavior test,
Thioflavin S，Aβ ELISA
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同济大学 硕士学位论文 目录
目录
第 1 章 引言 ............................................................................................................... 1 1.1 老年痴呆症的概述 .......................................................................................... 1 1.2 老年痴呆症的发病机制 .................................................................................. 1 1.2.1 Aβ毒性学说 ............................................................................................. 1 1.2.2 基因突变学说 ........................................................................................... 2 1.2.3 微管相关蛋白Tau异常学说 .................................................................... 2 1.3 老年痴呆症的治疗药物 .................................................................................. 3 1.4 老年痴呆症的小鼠模型 .................................................................................. 5 1.4.1 β-淀粉样蛋白沉积转基因小鼠模型 ....................................................... 5 1.4.2 转载脂蛋白E(Apo E)基因小鼠 ............................................................... 7 1.4.3 Tau蛋白沉积转基因小鼠模型 ................................................................ 7 1.5 老年痴呆症小鼠模型的行为学检测方法 ...................................................... 8 1.5.1 水迷宫实验 ............................................................................................... 8 1.5.2 Barnes迷宫实验 ....................................................................................... 9 1.5.3 条件性恐惧测试 ..................................................................................... 9 1.5.4 新物体识别实验 ....................................................................................... 9 1.5.5 T迷宫实验 .............................................................................................. 10 1.6 老年痴呆症小鼠模型的生化及病理检测指标 ............................................ 10 1.7 小结及本研究目的 ........................................................................................ 10 第 2 章 材料与方法 ................................................................................................. 12 2.1 材料与仪器 .................................................................................................... 12 2.1.1 实验动物 ................................................................................................. 12 2.1.2 实验仪器 ................................................................................................. 12 2.1.3 主要试剂及配方 ..................................................................................... 13 2.2 实验方法 ........................................................................................................ 14 2.2.1 APP(swe)/PSEN1dE9和Tg6799小鼠基因型的鉴定 .............................. 14 2.2.2 新物体识别实验 ..................................................................................... 15 2.2.3 Morris水迷宫 ......................................................................................... 17 2.2.4 动物脑组织的处理及病理染色 ............................................................. 18 2.2.5 脑组织Western blot .............................................................................. 19 2.2.6 脑组织和血清ELISA实验 ..................................................................... 19 2.3 统计数据分析 ................................................................................................ 20 第 3 章 结果 ............................................................................................................. 21 3.1 老年痴呆症转基因小鼠的鉴定 .................................................................... 21
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同济大学 硕士学位论文 目录
3.1.1 APP(swe)/PSEN1dE9和Tg6799小鼠基因型鉴定 .................................. 21 3.2 APP(swe)/PSEN1dE9和Tg6799老年痴呆症行为学检测 ............................. 22 3.2.1 不同月龄APP(swe)/PSEN1dE9和Tg6799小鼠的水迷宫实验 ................ 22
3.2.2 不同月龄APP(swe)/PSEN1dE9和Tg6799小鼠的新物体识别实验 ........ 23 3.3 APP(swe)/PSEN1dE9和Tg6799老年痴呆症小鼠脑组织生化指标检测 ..... 25 3.3.1 脑中APP含量的测定 ............................................................................. 25 3.3.2 额叶皮层和海马组织磷酸化Tau的检测 .............................................. 25 3.3.3 A?检测 ................................................................................................... 27 3.4 APP(swe)/PSEN1dE9和Tg6799老年痴呆症小鼠脑切片染色 ..................... 30 3.4.1 4G8免疫染色与Thioflavin S染色的对比 ............................................. 30 3.4.2 APP(swe)/PSEN1dE9和Tg6799小鼠脑内A??Thioflavin S染色 ............ 31 3.5 Tg6799老年痴呆症小鼠作为AD治疗药物筛选模型的应用 ..................... 34 第 4 章 总结与讨论 ............................................................................................... 35 致谢 ............................................................................................................................. 37 参考文献 ..................................................................................................................... 38 个人简历、在读期间发表的学术论文及研究成果 ................................................. 43
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