拟阿尔茨海默病小鼠Neprilysin功能及调控变化的研究

发布时间：2019-01-28 08:21

【摘要】：阿尔茨海默症(Alzheimers disease,AD)是以进行性记忆和认知功能损害为特征的神经退行性疾病,是威胁人类晚年健康的一大隐患,其主要病理特征之一为β-淀粉样蛋白(Ap)的聚集。NEP又称为中性内肽酶或脑啡肽酶(Neprilysin),由750个氨基酸组成。NEP是一种膜结合糖蛋白,包括一个短N-末端胞内区,跨膜区和胞外催化区,该催化区促进胞外寡钛(5kDa,如Aβ40and Aβ42peptides)疏水氨基酸残基端的水解。NEP在很多正常组织中均有表达,包括小肠和肾小管上皮细胞的刷状缘、中性粒细胞、胸腺细胞、肺、前列腺、睾丸和脑组织。人脑组织中NEP主要存在于神经元的胞膜,突触前和突触后膜,在黑质-纹状体通路及Aβ沉积的易感区(如海马)等脑区广泛分布,可以降解Ap的单体和多聚体。实验结果提示NEP结构和功能的变化和Aβ的聚集关系密切。本研究主要的目的为探索NEP在阿尔茨海默症发病中的变化规律,重点探讨NEP亚细胞定位及活性变化规律及与AD发病的关系。由于AD病人脑的研究一般都是疾病晚期,很难观察其发展过程。所以我们使用APP/PS1双转基因小鼠模型和正常野生型小鼠进行分组实验,而转基因小鼠也按照不同月龄分为不同的四组：3个月组,6个月组,9个月组,12个月组。首先采用Morris水迷宫实验,观察不同月龄的转基因小鼠的学习记忆情况,用来判断其AD的病情严重程度；之后对小鼠脑切片进行硫磺素染色和免疫组织化学染色,来检测Ap的具体沉积情况和NEP在大脑中的分布；之后使用新鲜的脑组织进行亚细胞分离,用提取液进行NEP的活性测试。实验结果显示,在转基因小鼠中,超过三个月龄后,都会有不同程度的Ap沉积,而且逐年增加。但是从水迷宫的实验来看,却是只有在年龄达到9个月时,才会出现AD特有的学习和记忆方面的障碍。而之后的活性测试则表明,NEP在细胞中的活性随月龄的增长出现先升高后下降的现象,其中在3-6个月时出现峰值,其他月龄时则和野生型小鼠保持相同。通过本次试验,证明了NEP能影响Aβ所引起的学习和记忆障碍,抑制AD的病情发展,其机制可能主要是Ap斑块的增加所引起NEP活性的上升,但在Ap沉积到一定数量时,反而抑制了NEP的活性,促使AD病情急剧加重,但是具体的机制还需要进一步的探索。
[Abstract]:Alzheimer's disease (Alzheimers disease,AD) is a neurodegenerative disease characterized by progressive memory and cognitive impairment. One of its main pathological features is the aggregation of 尾 -amyloid (Ap). NEP, also known as neutral endopeptidase or enkephalase (Neprilysin), is composed of 750 amino acids. NEP is a membrane-binding glycoprotein containing a short N-terminal intracellular region. The transmembrane and extracellular catalytic regions promote the hydrolysis of hydrophobic amino acid residues of extracellular oligotitanium (5 kDa, such as A 尾 40and A 尾 42peptides). NEP is expressed in many normal tissues, including brushes, neutrophils, and neutrophils in epithelial cells of small intestine and renal tubules. Thymocytes, lungs, prostate, testis and brain tissue. NEP mainly exists in the cellular membrane, presynaptic and postsynaptic membrane of neurons, and is widely distributed in the nigra striatum pathway and the susceptible areas of A 尾 deposition, such as the hippocampus, which can degrade the monomers and polymers of Ap. The results suggest that the changes of NEP structure and function are closely related to the aggregation of A 尾. The main purpose of this study was to explore the changes of NEP in Alzheimer's disease, and to explore the relationship between NEP subcellular localization and activity and the pathogenesis of AD. Because the brain of AD patients is usually late, it is difficult to observe its development. So we used the APP/PS1 double transgenic mice model and the normal wild type mice to carry on the grouping experiment, and the transgenic mice were divided into four groups according to different month age: 3 month group, 6 month group, 9 month group, 12 month group. First, Morris water maze test was used to observe the learning and memory of transgenic mice of different months of age to judge the severity of AD. The specific deposition of Ap and the distribution of NEP in the brain were detected by sulfur staining and immunohistochemical staining, and the subcellular separation of fresh brain tissue was carried out, and the activity of NEP was tested with the extract. The results showed that Ap deposition in transgenic mice increased year by year after three months of age. But from the water maze experiment, it is only at the age of nine months, the emergence of AD specific learning and memory disorders. The later activity tests showed that the activity of NEP in the cells increased first and then decreased with the increase of age, the peak value appeared at 3 to 6 months, and the other months were the same as those in the wild type mice. Through this experiment, it was proved that NEP can affect the learning and memory impairment induced by A 尾 and inhibit the development of AD. The mechanism may be the increase of NEP activity caused by the increase of Ap plaque, but when the Ap is deposited to a certain amount, On the contrary, the activity of NEP was inhibited, and the condition of AD was rapidly aggravated, but the specific mechanism needed to be further explored.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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