本文關(guān)鍵詞： 阿爾茨海默癥(AD) 硒酸鈉 Wnt/β-catenin信號通路 蛋白磷酸酯酶2A(PP2A) β-淀粉樣蛋白前體(APP) β-淀粉樣蛋白(Aβ)　出處：《深圳大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：阿爾茨海默癥(Alzheimer’s disease,AD)是一種與年齡相關(guān)的漸行性神經(jīng)退行性疾病,最初表現(xiàn)為進行性記憶力減退、獲得性知識喪失,最終發(fā)展為日常生活活動能力完全喪失,給社會和家庭帶來沉重負擔(dān)。越來越多的研究表明硒的日攝入量與AD的發(fā)病率密切相關(guān)。研究發(fā)現(xiàn)硒酸鈉能夠顯著降低微管相關(guān)蛋白tau病變模型鼠腦內(nèi)tau蛋白的過度磷酸化,減少神經(jīng)纖維纏結(jié),但是硒酸鈉對AD病理過程中Wnt/β-catenin信號通路的影響以及對淀粉樣蛋白前體(APP)的剪切過程的影響并不清楚。同時,AD病變過程與嗅球的關(guān)系、硒對AD鼠腦中嗅球病變的影響,目前報道甚少。本論文圍繞這兩方面開展了相關(guān)研究。本研究采用三轉(zhuǎn)基因AD模型鼠,從2月齡開始給予含6?g/ml硒酸鈉的飲用水,給藥10個月后檢測其海馬區(qū)的病理指標。研究發(fā)現(xiàn),12月齡AD模型鼠海馬區(qū)出現(xiàn)大量β-淀粉樣蛋白(Aβ)及神經(jīng)元凋亡,并且出現(xiàn)Wnt/β-catenin信號通路活性下調(diào)。給予硒酸鈉的AD模型鼠,其海馬區(qū)神經(jīng)元突觸密度增加,β-淀粉樣蛋白減少,神經(jīng)元凋亡降低。進一步研究發(fā)現(xiàn):硒酸鈉顯著激活A(yù)D模型鼠腦內(nèi)蛋白磷酸酶(PP2A)的活性、降低糖元合成酶激酶3β(GSK3β)的磷酸化,提高β-鏈蛋白(β-catenin)的表達水平,從而激活Wnt/β-catenin信號通路及其下游靶基因的轉(zhuǎn)錄,使基因c-myc、survivin、TXNRD2表達水平上調(diào),基因BACE1的表達水平下調(diào)。AD模型鼠腦內(nèi)APP在其第668位的蘇氨酸殘基的磷酸化(APP-pT668)易被BACE1識別并剪切,造成Aβ的產(chǎn)生增加,本研究發(fā)現(xiàn)AD模型鼠硒酸鈉處理組APP-pT668的表達水平顯著降低,降低Aβ的產(chǎn)生。以上結(jié)果表明Wnt/β-catenin信號通路對阻止AD的病理變化具有重要作用,硒酸鈉有可能是治療AD的一種潛在藥物。AD的早期病理變化中嗅覺功能受損,為了探究硒酸鈉對嗅球早期病理變化的影響,本研究給予2月齡三轉(zhuǎn)基因AD模型鼠3?g/ml硒酸鈉飲用水,飼養(yǎng)4個月后檢測相關(guān)指標。結(jié)果發(fā)現(xiàn),硒酸鈉顯著提高小鼠的空間記憶能力,相比野生型小鼠,AD組嗅球內(nèi)Aβ及Tau-pS231的表達顯著升高,硒酸鈉處理組AD模型鼠嗅球內(nèi)二者的表達水平顯著降低。分別取野生型小鼠、AD模型鼠及硒酸鈉飼養(yǎng)AD組小鼠的嗅球組織,從中提取總RNA進行轉(zhuǎn)錄組測序和生物信息學(xué)分析,發(fā)現(xiàn)與野生型小鼠相比,AD小鼠嗅球中Grin2a、Grin2b、Creb1、Gng13及Chrm2基因表達水平降低,Gfap基因表達水平升高;給予硒酸鈉的AD模型小鼠,嗅球中顯著上調(diào)的基因有Grin2a、Grin2b、Creb1、Gng13及Chrm2,下調(diào)基因有Gfap,硒酸鈉給藥組逆轉(zhuǎn)了AD模型鼠嗅球內(nèi)上述基因的表達水平。采用Q-PCR及Western blotting驗證了與突觸相關(guān)的基因Grin2a和Grin2b與轉(zhuǎn)錄組顯示的結(jié)果一致。為了在細胞水平上驗證這一結(jié)果,用0.1?M硒酸鈉處理從AD小鼠嗅球中分離培養(yǎng)的原代神經(jīng)元,與從野生型小鼠嗅球中分離培養(yǎng)的原代神經(jīng)元相比較,發(fā)現(xiàn)AD組嗅球內(nèi)神經(jīng)元突觸素蛋白顯著下降,硒酸鈉處理AD組,突觸素的表達增加。綜上,硒酸鈉能夠激活A(yù)D模型鼠腦內(nèi)降低的Wnt/β-catenin信號通路,抑制APP剪切形成Aβ、減少神經(jīng)元的凋亡。同時,硒酸鈉降低嗅球內(nèi)Aβ的產(chǎn)生及tau蛋白的過度磷酸化,提高嗅球中Grin2a和Grin2b基因和蛋白的表達,并且提高突觸素蛋白的表達。在已有硒酸鈉抑制AD病理特征的基礎(chǔ)上,進一步揭示了硒酸鈉對AD上游信號通路和早期病變組織的作用和機理。
[Abstract]:Blzheimer ("Alzheimer s disease, AD) is more of a neurodegenerative disease associated with age, initially characterized by progressive loss of memory, loss of acquired knowledge, and ultimately the development of activities of daily living completely lost, bring heavy burden to society and family. More and more studies show that the incidence of selenium on intake and rate of AD are closely related. The study found that sodium selenite can significantly reduce tau hyperphosphorylation of microtubule associated protein tau in brain of rat model, reduce neurofibrillary tangles, but the sodium selenite AD pathological effects of Wnt/ during beta -catenin signaling pathway and the amyloid precursor protein (APP) effect the shearing process is not clear. At the same time, the relationship between AD and pathological changes of the olfactory bulb, effects of selenium on AD in rat brain in the olfactory bulb lesion, rarely reported. This paper carried out the research on the two aspects Study. This study used three AD transgenic mouse model, starting from 2 month old to 6 with g/ml? Sodium selenite in drinking water, detection of pathological index in their hippocampus after 10 months of administration. The study found that 12 month old of the AD model in hippocampus of the emergence of a large number of amyloid beta (A beta) and neuronal apoptosis, and downregulation of Wnt/ beta -catenin signaling pathway activity. AD model rats were given sodium selenite, the hippocampal synaptic density increased, amyloid beta protein decreased, apoptosis of neurons decreased. Further studies showed that sodium selenite significantly activated AD model rat brain protein phosphatase (PP2A) activity, reduced glycogen synthase kinase 3 beta (GSK3 beta) phosphorylation, improved beta chain protein (beta -catenin) expression levels and transcriptional activation of Wnt/ -catenin signaling pathway and its downstream target genes, the genes c-myc, survivin, TXNRD2 expression level increased, the expression level of BACE1 gene down regulated. AD APP in the brain of rat model of threonine residues 668th phosphorylation of BACE1 (APP-pT668) is easily identified and cut, resulting in the increase of A beta, the study found that the AD rat model of sodium selenite treatment group the expression level of APP-pT668 decreased significantly, decreased the production of A 3. The above results showed that Wnt/ beta -catenin the signal pathway plays an important role in the pathological changes of block AD, sodium selenite may be the early pathological changes in olfactory function damaged a potential drug for treatment of.AD AD, in order to explore the effect of sodium selenate on early pathological changes of the olfactory bulb, this study gives 2 month old three transgenic rat model of AD 3? G/ml sodium selenite in drinking water. Detection of relevant indicators after 4 months of feeding. The results showed that sodium selenite significantly improve the spatial memory ability of mice, compared with wild type mice, AD group in the olfactory bulb of the expression of A beta and Tau-pS231 increased significantly, sodium selenite treated group AD rats in the olfactory bulb The expression level of two was significantly reduced. Were collected from wild type mice, the olfactory bulb tissue in AD rats and sodium selenite feeding AD mice, extraction of total RNA from in transcriptome sequencing and bioinformatics analysis, found that compared with wild type mice, Grin2a AD mice, the olfactory bulb in Grin2b, Creb1, Gng13 expression and the level of Chrm2 gene decreased, the expression level of Gfap gene increased; AD mice given sodium selenite, significantly increased in the olfactory bulb gene Grin2a, Grin2b, Creb1, Gng13 and Chrm2, were down regulated Gfap, sodium selenite reversed the expression level of AD rats in the olfactory bulb of the gene delivery by Q-PCR group. Western and blotting show and synapse related genes Grin2a and Grin2b and transcription group showed the consistency of the results. In order to verify the results, at the cellular level by 0.1? M sodium selenite treatment cultured primary neurons from the olfactory bulb of AD mice, and from the wild Cultured primary neurons in the olfactory bulb type mice compared to AD group of neurons in the olfactory bulb of synaptophysin decreased significantly in treatment group AD of sodium selenite, increase the expression of synaptophysin. Therefore, sodium selenite can activate Wnt/ beta -catenin signaling pathway in AD rat brain decreased, the inhibition of APP formation of shear A beta. Reduce neuronal apoptosis. At the same time, sodium selenite reduced phosphorylation in the olfactory bulb of A beta and tau protein, enhance the expression of Grin2a and Grin2b in the olfactory bulb of gene and protein, and increase the expression of synaptophysin. Inhibition of AD based histopathological features in existing sodium selenite, and further reveal the mechanism of selenium AD sodium on the upstream signaling pathways and early lesions.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16;R-332

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