口服甲醇建立的小鼠和恒河猴AD动物模型
文献类型：学位论文
| 作者 | 杨美凤 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 胡新天 |
| 关键词 | 甲醇 甲醛 阿尔兹海默病（AD) 记忆损伤 tau蛋白磷酸化 脑脊液 空间延迟任务 |
| 其他题名 | Methanol feeding lead to AD like model in mice and monkeys |
| 学位专业 | 神经生物学 |
| 中文摘要 | 第一部分：口服甲醇引起的AD动物模型 这一章首先对前人建立的阿尔兹海默（Alzheimer’s disease, AD）模型进行了综述，之后汇报了作者的博士学位论文研究工作。实验主要采用口服甲醇的方式建立小鼠AD动物模型，之后通过行为学、免疫组化等方式检测模型的有效性。阿尔茨海默病是一种由神经退行性紊乱引起的老龄化痴呆症，它与大脑内淀粉样沉淀（Amyloid Plaques）的形成及神经纤维缠结（Neurofibral tangles, NFTs）有关。本实验中，把小鼠分为三组每组9只分别口服清水、浓度为2%甲醇和3.8%甲醇，连续服用6周，停药十天后进行Y迷宫和嗅觉记忆实验，发现小鼠的记忆能力受到损伤，具体表现在小鼠无法识别新异臂和柠檬气味。之后脑组织免疫组化的实验中显示口服甲醇组小鼠海马区CA1的tau蛋白有明显的磷酸化，并且CA1区细胞有明显的凋亡。由于甲醇在体内可以被代谢为甲醛和甲酸，无法确认脑损伤由哪种物质引起，因此进行了对小鼠胚胎皮层细胞和N2a细胞两个体外实验，将两种类型的细胞分别用甲醇、甲醛和甲酸进行培养，结果表明甲醛培养的小鼠胚胎大脑皮层细胞的细胞骨架蛋白解体严重，且甲醛引起的N2a细胞tau蛋白磷酸化程度远高于甲醇和甲酸组，实验中甲醇很可能代谢成甲醛之后通过海马中相关的Tau蛋白磷酸化致使细胞凋亡来损伤小鼠学习记忆功能。由以上的行为测试、免疫组化实验和体外实验证实我们建立了一个建立在tau蛋白致病说基础上非常类似于AD的小鼠动物模型。 关键词：甲醇，甲醛，阿尔兹海默病（AD），认知损伤，tau蛋白磷酸化 第二部分：口服甲醇建立的恒河猴AD模型 本章首先对猕猴建立的AD模型做了综述，之后报道了采用口服甲醇方式建立的猕猴AD动物模型，之后通过行为学，免疫组化等方式检测模型的有效性。 选取年龄在3-4岁的4只猕猴，实验前给猕猴做空间延迟任务，之后给甲醇，每日以2ml/kg的剂量溶解在水中配成终浓度3%的溶液给猕猴自由饮用，给药一年半后抽取脑脊液检测tau蛋白磷酸化程度，期间测定空间延迟任务成绩，给药特定时间后停药3个月，6个月再次测定空间延迟任务，选取两只猕猴灌流，取脑切片检测脑内Aβ沉淀情况和tau蛋白磷酸化情况。实验结果表明给药一个月后猕猴的空间延迟任务有明显下降，停药3个月和6个月后成绩未恢复，表明甲醇引起的记忆下降是不可逆的、稳定的，给药一年半后脑脊液中出现明显的tau蛋白磷酸化，脑切片中有明显的Aβ沉淀和tau蛋白磷酸化，以上行为学和病理学检测结果表明此模型可作为有效的AD模型。 |
| 英文摘要 | Part 1: Methanol feeding lead to AD like tauopathy in mice Alzheimer's disease (AD) is a progressive neurodegenerative disorder causing age-related dementia. It is associated with genetic factors leading to amyloid plaque formation and neurofibillary tangles within the brain. A proper animal model is important for Alzheimer's disease (AD) research. Recent studies have established a link between the metabolite formaldehyde (FA) and AD pathologies, including one of the AD's most important characteristics - Tau hyperphosphorylation. In the present study, mice were fed with methanol as a FA precursor to develop a new AD animal model. Three groups of mice (N = 9) were given either water as a control or a methanol solution (concentrations of 2% and 3.8%) for a six week period. AD-dementia like symptoms were confirmed in the mice with Y-maze and olfactory memory tests in order to assess spatial recognition and olfactory memory, respectively. Then tau hyperphosphorylation, amyloid plague formation and neuronal apoptosis of hippocampus CA1 were measured in the mouse brains. In contrast to the control group, the methanol-fed groups were found to have a level of memory impairment; they failed to recognize the novel arm in the Y-maze apparatus and failed to establish recognition of a lemon odor in the olfactory test. Immunohistochemistry experiments found increased neuronal Tau phosphorylation in the hippocampus and increased cellular apoptotic markers in hippocampal CA1 neurons (approx. 10% of neurons displayed chromatin condensation) in the methanol-fed groups, but were unable to find changes in amyloid-plaque formation. Two additional in vitro experiments were performed in mouse embryonic cerebral cortical neurons and N2a cells to evalutate which methanol metabolite caused AD associated changes (e.g. tau phosphorylation). The in vitro investigations found that FA, not methanol nor formic acid, significantly induced microtubule disintegration and Tau protein hyperphosphorylation. All told, the behavioral tests, immunohistochemical analysis, and in vitro experiments indicated the FA impaired memory through hippocampal related tau-phosphorylation. Furthermore, these findings suggested that the methanol-fed mouse model developed here is a new AD animal model based on tauopathies, especially in the Tau-hyperphosphorylation dependent loss of microtubules in tangle formation. Keywords: methanol, formaldehyde, Alzheimer's disease, cognitive impairment, Tau hyperphosphorylation. Part 2: Methanol feeding lead to AD model in monkeys Four monkeys (N = 4) were fed with methanol solution (dose 2 ml/kg, concentration: 3% diluted in water) over a one month period. To confirm AD-dementia like symptoms in the monkeys, variable spatial delayed tasks (VSTD) were used to determine whether the working memory was impaired in each monkey before and after the chemical feed. Then, Aβdeposits were measured in the monkeys’ brains post-mortem as well as the Tau hyperphosphorylation in the CSF (Cerebral Spinal Fluid). The monkeys failed to remember which side of the apparatus contained the food reward during the variable spatial delayed tasks （VSTD） after the methanol-feeding regimen in contrast to the baseline before the methanol-feeding. The monkeys were also found to have a similar correct response rate as that found immediately following the methanol-feeding regimen after a 3 month or a half year recovery period. This indicated that the level of memory impairment was stable over an extended period. Furthermore, immunohistochemistry found increased neuronal Tau phosphorylation in the CSF and Aβdeposits as well as tau phosphorylation in the monkeys’ brains. The findings in the behavior tests that monkeys given methanol had dysfunctional working memory as well as the findings of Aβdeposits and tau phosphorylation in the brain sections and CSF Tau phosphorylation suggested that the methanol-fed monkey model developed here is a new AD-like animal mo |
| 语种 | 中文 |
| 公开日期 | 2014-07-01 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7915]  |
| 专题 | 昆明动物研究所_神经系统编码 |
| 推荐引用方式 GB/T 7714 | 杨美凤. 口服甲醇建立的小鼠和恒河猴AD动物模型[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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