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標題: | 慢性暴露大氣懸浮微粒對阿茲海默症三基因轉殖小鼠相關病徵之影響 Effects of chronic exposure to ambient particles on pathology of Alzheimer’s disease in 3xTg-AD mice. |
作者: | Ching-Chou Hsu 許景洲 |
指導教授: | 鄭尊仁(Tsun-Jen Cheng) |
關鍵字: | 大氣細懸浮微粒,阿茲海默症,空間記憶功能缺陷,神經元細胞死亡,自噬作用, ambient fine particles,Alzheimer’s disease,cognitive deficit,neuron death,autophagy, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 隨著民眾健康意識提升,空氣汙染議題日益受到重視,暴露大氣懸浮微粒(Particulate matter, PM)會對健康產生許多不良效應,過去許多研究已經證實了微粒暴露與心血管、呼吸道疾病之間的致病與機轉。近年來有研究指出,暴露小粒徑的微粒,如細懸浮微粒PM2.5,會影響中樞神經系統(Central nervous system, CNS),造成阿茲海默症(Alzheimer’s disease, AD)等之神經退化性疾病(Neurodegenerative disease)。 AD於病理學上主要有兩大特徵:第一種為由乙型類澱粉蛋白(Beta-amyloid, Aβ)聚集沉積而成的類澱粉蛋白斑塊(Aβ plaque);第二種為Tau蛋白因過度磷酸化而形成的神經纖維纏結(Neurofibrillary tangles, NFTs),這兩種病徵會傷害神經元細胞,造成細胞死亡,最終導致患者認知功能喪失。除此之外,研究指出氧化壓力、微膠細胞活化所導致的發炎反應上調也對神經元細胞死亡有著一定的貢獻。 本研究使用台北空氣汙染暴露系統(Taipei air pollution exposure system, TAPES)進行暴露實驗,TAPES能直接抽取外界大氣中的微粒,輸入至暴露腔內,提供一非濃縮、符合真實大氣環境之暴露條件。此外,本研究使用6個月大的阿茲海默症三基因轉殖小鼠當作實驗動物,進行為期5個月的慢性全身連續性呼吸暴露,由於此品系小鼠能隨著年齡發展出AD相關的病徵,適合用來探討暴露PM後是否會加速小鼠腦中AD相關病徵之變化。 暴露結束後,對小鼠進行了莫氏水迷津行為試驗(Morris water maze),以檢測其空間學習與記憶能力,並於行為試驗結束後進行犧牲。鼠腦會先進行核磁共振成像(Magnetic Resonance Imaging, MRI),並對大腦各區的體積進行定量。影像定量後,半腦的腦組織會細分為皮質(Cerebral cortex)與海馬迴(Hippocampus),並以西方墨點法(Western blot)測量AD特異性指標Aβ42與p-Tau、活化微膠細胞指標CD11b以及自噬作用指標LC3B的蛋白表現量;剩餘半腦的腦組織則進行石蠟包埋與切片染色,透過蘇木精-伊紅染色(Hematoxylin and eosin stain, H E stain)切片對神經元細胞進行計數,並以免疫組織化學染色(Immunohistochemistry stain, IHC stain)切片對AD特異性指標Aβ42、p-Tau、氧化壓力指標丙二醛(Malondialdehyde, MDA)、細胞凋亡酶Caspase-3以及自噬作用蛋白LC3的陽性細胞比例進行計數。 本研究小鼠暴露之PM2.5平均質量濃度為13.85 µg/m3。莫氏水迷津結果顯示在知識採集階段,兩組在各項參數皆無顯著差異,但在空間探索實驗,暴露組在目標象限平台停留時間顯著低於暴露組(p<0.05),其餘參數皆無顯著差異。神經影像學MRI分析結果顯示,兩組小鼠大腦各腦區之體積皆無顯著差異。阿茲海默症相關指標蛋白質定量分析結果顯示,暴露組大腦皮質有四項指標高於控制組(Aβ42 oligomer、 p-Tau、CD11b、LC3B),其中,Aβ42 oligomer蛋白表現量達到統計上之顯著差異(p<0.05),而海馬迴的各項指標皆無顯著差異。p-Tau蛋白表現量在各腦區皆無顯著差異。切片染色分析結果,H E staining切片顯示暴露組大腦皮質與內嗅皮質的神經元細胞數目顯著低於控制組(p<0.05),海馬迴則無顯著差異。IHC staining切片結果顯示,各項指標於各腦區皆無顯著差異。 我們的研究結果顯示慢性呼吸暴露大氣懸浮微粒會誘導小鼠大腦皮質中阿茲海默症特異性指標Aβ42 oligomer的上升與神經元細胞數目減少,導致小鼠的空間記憶能力下降,提供了微粒暴露加速阿茲海默症相關病徵形成的可能機制。 Previous studies have confirmed that particulate matter (PM) exposure can induce cardiovascular and respiratory disease. In recent years, PM2.5 has been implicated in the development of neurodegenerative disorders including Alzheimer's disease (AD). There are two specific markers in AD pathology including plaques formed by beta-amyloid (Aβ) accumulation and neurofibrillary tangles (NFTs) formed by aggregation of phosphorylated tau protein. These two markers can damage neuron cells, causing cell death and cognitive deficit. Moreover, oxidative stress and inflammation induced by activated microglia may contribute to AD pathology. We hypothesized that exposure to PM could accelerate AD-like pathology and cause cognitive deficit. To address this, we exposed 6-month-old female triple-transgenic AD mice (3xTg-AD mice), which can develop AD-like pathology, for 5 months to PM by inhalation. Using Taipei Air Pollution Exposure System (TAPES), the TAPES is designed to directly introduce the ambient particles to the exposure chamber, provide a continuous, real world and non-concentrated particle exposure. After exposure, Morris Water Maze (MWM) was used to assess the spatial learning and memory in mice. Magnetic Resonance Imaging (MRI) was used to quantify the brain volume in mice. The cerebral cortex and hippocampus were collected for analysis. Western blot was used to determine the expression of AD specific markers (Aβ42 and p-Tau protein), the marker of activated microglia (CD11b protein) and the marker of autophagy (LC3B protein). The neuronal cells and IHC-positive cells (Aβ42, p-Tau, CD11b, MDA, LC3 and caspase-3) were counted in brain slices. The average concentration of PM2.5 during the exposure was 13.85 µg/m3. While the control and exposure mice had learning effects in the escape latency, the exposed mice showed a significantly decreased spatial memory in time spent in platform quadrant (p<0.05). Western blot showed that the three markers (Aβ42 oligomer, p-Tau and CD11b) in exposure group were higher than controls in cerebral cortex, especially Aβ42 oligomer (p<0.05). The number of neuronal cells in cerebral cortex and entorhinal cortex were significantly decreased in exposure group (p<0.05). IHC-positive cells were not statistically significant between the control and exposure groups. Our study indicated that exposure to PM by inhalation could accelerate AD-like pathology in 3xTg-AD mice in cerebral and entorhinal cortex, causing neuronal death and spatial memory deficit. The study results also support the previous epidemiological findings on the associations between PM and AD. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50032 |
DOI: | 10.6342/NTU202002982 |
全文授權: | 有償授權 |
顯示於系所單位: | 環境與職業健康科學研究所 |
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