【作者】 方瑾；

【导师】 秦川； 张连峰；

【作者基本信息】 中国协和医科大学 ， 病理与病理生理学， 2008， 硕士

【摘要】 London/Swedish APP双突变转基因小鼠模型的建立和初步表型分析阿尔茨海默病（Alzheimer’s disease,AD）是一种常见的中枢神经系统退行性变性疾病,其主要临床表现为进行性记忆减退和认知障碍。AD的主要神经病理学特征为过度的细胞外β淀粉样蛋白（amyloidβprotein,Ap）沉淀、细胞内神经纤维缠结增多、Tau蛋白的过度磷酸化、突触密度减少、神经元细胞减少等。为了更加深入的探究AD的发病机制,研究人员建立了一系列的动物模型作为研究工具。淀粉样蛋白前体蛋白（The amyloid precursor protein,APP）在AD发病机制中起重要作用,APP经过剪切加工后的Aβ是AD患者脑内老年斑的主要成分,而点突变的APP则是早发性AD（early onset of familialAlzheimer’s disease,FAD）的主要病因。目前国内外已经建立了多种APP突变型转基因小鼠模型如PDAPP转基因小鼠模型,APP23转基因小鼠模型等,但是大多数模型出现病理及行为学改变的时间比较滞后,通常在六月龄以后才出现上述改变。因此我们建立了London/Swedish APP双突变转基因小鼠,使小鼠能够比其它APP转基因动物更早的出现病症,提高使用模型进行研究时工作效率。本研究主要包括以下三个部分的实验内容:1.London/Swedish hAPP双突变转基因载体及小鼠的制备:将London/Swedish APP双突变基因插入到PDGF启动子下游,构建转基因表达载体,通过显微注射法建立APP695^{V652i/K596N/M597L}转基因C57BL/6J小鼠。2.分子生物学水平及病理学分析:采用PCR技术鉴定APP695^{V652I/K596N/M597L}转基因小鼠的基因表型,鉴定出四只Founder鼠。进一步用RT-PCR和Western blotting检测hAPP695^{V652I/K596N/M597L}基因的表达,筛选并建立了2高表达的系APP695^{V652I/K596N/M597L}转基因小鼠。免疫组织化学显示在海马区双突变APP695^{V652I/K596N/M597L}转基因小鼠出现的阳性细胞数目较单突变APP695^*（652I）转基因小鼠及野生小鼠明显增多。常规的病理学检查转基因小鼠脑组织未发现异常。3.行为学检测:采用水迷宫检测系统对APP695^{V652I/K596N/M597L}转基因小鼠的行为学改变进行鉴定。结果显示,三月龄时APP695^{V652I/K596N/M597L}转基因小鼠,单突变APP695^V652I转基因小鼠及野生小鼠在经过Morris水迷宫隐蔽平台实验时,APP695^{V652I/K596N/M597L}转基因小鼠寻找到隐蔽平台的潜伏期比APP695^V652I转基因小鼠及野生小鼠明显延长。探索试验发现APP^{V652I/K596N/M597L}转基因小鼠比APP^V652I转基因小鼠及野生小鼠穿越目标象限的次数明显减少。综上所述,APP695^{V652I/K596N/M597L}转基因小鼠较APP695^V652I转基因小鼠更早的表达hAPP,出现学习认知能力障碍的时间明显提前,说明我们成功建立APP695^{V652I/K596N/M597L}转基因小鼠阿尔茨海默病模型,为研究AD发病机制和药物研发提供了有价值的研究工具。更多还原

【Abstract】 Alzheimer’s disease（AD） is a common neurodegenerative disease, which is characterized clinically by a progressive loss of memory and cognitive impairment.Its main neuropathological features include accumulation of extracellular amyloid or senile plaques,diffuse loss of neurons in the hippocampus and neocortex,decrease of synapse density neurofibrillary tangles,excess phosphorylation of tau protein.For penetrating investigating the pathogenesis of AD,researchers have constructed a serial of animal models as research tool.The amyloid precursor protein takes a central position in AD pathogenesis:APP processing generates theβ-amyloid（Aβ） peptides,which are deposited as the amyloid plaques in brains of AD individuals;Point mutations and duplications of APP are causal for a subset of early onset of familial Alzheimer’s disease.At present,There are a certain number of mutation APP mice models,such as PDAPP transgenic mice model,APP 23 transgenic mice model et al.However,most of these models have appeared the pathology and ethology changes after six months.We have constructed and established the APP695^{V652I/K596N/M597L} transgenic mice model of Alzheimer’s disease.This kind of model can show up disorders earlier,elevate the utilization efficiency of model.The main works were included below.1.The construction of the London/Swedish hAPP mutation gene transgenic vector and the establishment of transgenic mice:The London/Swedish hAPP mutation transgenic vector was constructed by inserting the APP^{695V652I/K596N/M597L} gene into the downstream of PDGF promoter.The transgenic mice were generated for the APP^{695V6521/K596N/M597L} gene using microinjection method.2.Genotyping and pathological analysis:PCR was used to genotype the potential transgenic mice and 4 APP695^{V652I/K596N/M597L} founders were identified.The expression levels of the mutated gene were determined with the RT-PCR and Western blotting and 2 lines of APP695^{V652I/K596N/M597L} transgenic mice with high expression of the target gene were screened.The transgenic hAPP protein was localized in brain tissue immunohistochemistry.Compared with wild type mice and the APP695^V652I transgenic mice,more positive cells in the hippocampus was observed in the APP695^{V652I/K596N/M597L} brain.3.Behavioral test:The behavioral tests were examined by the water maze trials.We found that APP695^{V652I/K596N/M597L} transgenic mouse showed the longest average latency to find the hidden platform in the water maze than the APP695^V652I transgenic mouse and wild type mouse. APP695^{V652I/K596N/M597L} transgenic mouse have the less annulus crossing index than the other group in the probing test. Therefore,The APP695^{V652I/K596N/M597L} transgenic mouse showed the earlier pathological changes and spatial memory deficits compared with that of the APP695^V652I transgenic mouse and wild type mouse.It suggested that we have established an AD animal model successfully,and The APP695^{V652I/K596N/M597L} transgenic mouse is a useful AD animal model.更多还原