【摘要】：阿爾茨海默癥(Alzheimers disease,AD)是以進(jìn)行性記憶和認(rèn)知功能損害為特征的神經(jīng)退行性疾病,是威脅人類晚年健康的一大隱患,其主要病理特征之一為β-淀粉樣蛋白(Ap)的聚集。NEP又稱為中性內(nèi)肽酶或腦啡肽酶(Neprilysin),由750個氨基酸組成。NEP是一種膜結(jié)合糖蛋白,包括一個短N(yùn)-末端胞內(nèi)區(qū),跨膜區(qū)和胞外催化區(qū),該催化區(qū)促進(jìn)胞外寡鈦(5kDa,如Aβ40and Aβ42peptides)疏水氨基酸殘基端的水解。NEP在很多正常組織中均有表達(dá),包括小腸和腎小管上皮細(xì)胞的刷狀緣、中性粒細(xì)胞、胸腺細(xì)胞、肺、前列腺、睪丸和腦組織。人腦組織中NEP主要存在于神經(jīng)元的胞膜,突觸前和突觸后膜,在黑質(zhì)-紋狀體通路及Aβ沉積的易感區(qū)(如海馬)等腦區(qū)廣泛分布,可以降解Ap的單體和多聚體。實驗結(jié)果提示NEP結(jié)構(gòu)和功能的變化和Aβ的聚集關(guān)系密切。 本研究主要的目的為探索NEP在阿爾茨海默癥發(fā)病中的變化規(guī)律,重點(diǎn)探討NEP亞細(xì)胞定位及活性變化規(guī)律及與AD發(fā)病的關(guān)系。由于AD病人腦的研究一般都是疾病晚期,很難觀察其發(fā)展過程。所以我們使用APP/PS1雙轉(zhuǎn)基因小鼠模型和正常野生型小鼠進(jìn)行分組實驗,而轉(zhuǎn)基因小鼠也按照不同月齡分為不同的四組：3個月組,6個月組,9個月組,12個月組。首先采用Morris水迷宮實驗,觀察不同月齡的轉(zhuǎn)基因小鼠的學(xué)習(xí)記憶情況,用來判斷其AD的病情嚴(yán)重程度；之后對小鼠腦切片進(jìn)行硫磺素染色和免疫組織化學(xué)染色,來檢測Ap的具體沉積情況和NEP在大腦中的分布；之后使用新鮮的腦組織進(jìn)行亞細(xì)胞分離,用提取液進(jìn)行NEP的活性測試。 實驗結(jié)果顯示,在轉(zhuǎn)基因小鼠中,超過三個月齡后,都會有不同程度的Ap沉積,而且逐年增加。但是從水迷宮的實驗來看,卻是只有在年齡達(dá)到9個月時,才會出現(xiàn)AD特有的學(xué)習(xí)和記憶方面的障礙。而之后的活性測試則表明,NEP在細(xì)胞中的活性隨月齡的增長出現(xiàn)先升高后下降的現(xiàn)象,其中在3-6個月時出現(xiàn)峰值,其他月齡時則和野生型小鼠保持相同。 通過本次試驗,證明了NEP能影響Aβ所引起的學(xué)習(xí)和記憶障礙,抑制AD的病情發(fā)展,其機(jī)制可能主要是Ap斑塊的增加所引起NEP活性的上升,但在Ap沉積到一定數(shù)量時,反而抑制了NEP的活性,促使AD病情急劇加重,但是具體的機(jī)制還需要進(jìn)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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R749.16

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