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Title: | 以自發性高血壓大鼠亞慢性暴露大氣微粒對神經毒性的影響 Neurotoxicity induced by subchronic inhalation exposure to ambient particulate matter in Spontaneously Hypertensive Rats |
Authors: | Pei-Jui chai 柴培睿 |
Advisor: | 鄭尊仁(Tsun-Jen Cheng) |
Keyword: | 大氣細懸浮微粒,神經毒性,氧化壓力,Tau蛋白,莫氏水迷津, ambient particles,neurotoxicity,oxidative stress,Tau protein,Morris Water Maze, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 許多流行病學與毒理學研究皆證實暴露於大氣懸浮微粒(Particulate matter, PM)會增加中樞神經的氧化壓力,進而引起神經退行性疾病,包括阿茲海默症(Alzheimer’s disease, AD),阿茲海默症在腦中的主要病理特徵,是由乙型類澱粉蛋白(Amyloid-beta, Aβ)沈積所形成老年斑塊(Senile plaques),以及由過度磷酸化Tau蛋白(Hyperphosphorylated Tau protein)聚集所組成神經纖維纏結(Neurofibrillary tangles, NFTs),另外有研究也指出氧化壓力會誘導Tau蛋白過度磷酸化以及NFTs形成,進而引起阿茲海默症,此外,高血壓被認為是阿茲海默症的危險因子之一,因此本研究使用自發性高血壓動物模式,並假設暴露於懸浮微粒會加速自發性高血壓大鼠(Spontaneously hypertensive rats, SHR)在腦中形成阿茲海默症病徵,因此本研究欲探討亞慢性暴露大氣懸浮微粒後,氧化壓力指標和阿茲海默症病徵在腦中的變化。
本研究以8周大的SHR使用台北空氣污染暴露系統(Taipei Air Pollution Exposure System, TAPES)進行三個月與六個月的全身性呼吸暴露,此系統可提供動物全身暴露於連續、非濃縮的真實大氣懸浮微粒,暴露三個月後進行莫氏水迷津(Morris water maze, MWM),MWM完成後,再進行暴露,待總暴露時間到達六個月,暴露結束後大鼠進行犧牲,取出腦組織,細分為嗅球(Olfactory bulb)、小腦(Cerebellum)、海馬迴(Hippocampus)以及皮質(Cerebral cortex)四個腦區,針對四個不同腦區測定氧化壓力指標丙二醛(Malondialdehyde, MDA)以及阿茲海默症病徵指標總Tau蛋白(Total Tau protein, t-Tau)和磷酸化Tau蛋白(Phosphorylated Tau protein, P-tau)。 本研究大鼠暴露前三個月PM2.5之平均質量濃度為8.6 μg/m3,暴露後三個月PM2.5之平均質量濃度為10.8 μg/m3,行為實驗結果顯示知識採集階段和空間探索實驗在控制組和暴露組之間皆無顯著差異,而控制組以及暴露組有著一條合理的學習曲線;在氧化壓力指標MDA的部分,暴露三個月在四個腦區皆無顯著差異,暴露六個月在嗅球、海馬迴及皮質則顯著上升;在阿茲海默症指標Tau蛋白的部分,暴露三個月t-Tau和p-Tau蛋白表現量在嗅球有顯著上升,而暴露六個月在四個腦區皆無顯著差異;在組織病理切片的部分,肺部在暴露三和六個月的控制組與暴露組皆無明顯的病理變化,而腦部暴露六個月的控制組與暴露組也無明顯的病理變化。 研究結果顯示亞慢性呼吸暴露大氣懸浮微粒會誘導腦中的氧化壓力上升與Tau蛋白增加,然而三個月大氣懸浮微粒暴露並未對SHR的空間學習及記憶功能造成影響,本研究說明呼吸暴露大氣懸浮微粒造成中樞神經產生毒性的可能機制,但本實驗所探討與中樞神經毒性相關的指標仍不足,因此未來的研究需要更進一步來釐清大氣懸浮微粒對中樞神經的影響。 Recently, many studies have shown that exposure to particulate matter (PM) may induce oxidative stress in the central nervous system (CNS) and contribute to neurodegenerative diseases, such as Alzheimer’s disease (AD). The major pathological characteristics of AD brain are senile plaque of amyloid-beta (Aβ) and neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein. Some findings have also indicated the oxidative stress would induce Tau protein hyperphosphorylation and NFTs formation and contribute to AD. Moreover, hypertension is a well-known risk factor for Alzheimer’s disease. Thus, we hypothesized that ambient particulate matter could accelerate Alzheimer’s disease-like effects in spontaneously hypertensive rats (SHR). In the experiment, we use SHR to explore the relationship between PM exposure and markers of oxidative stress and markers of AD through subchronic inhalation. 8-week-old male SHR were exposed to continuous, non-concentrated, real world PM2.5 using Taipei Air Pollution Exposure System (TAPES) for 3 and 6 months. After 3 months exposure, Morris Water Maze (MWM) was conducted. Afterwards, the rats were proceeded an another 3 months exposure of PM2.5 for a total of 6 months. After the exposure, the brain tissue, including olfactory bulb, cerebellum, hippocampus and cerebral cortex, were collected. The level of malondialdehyde (MDA), maker of oxidative stress, was determined by LC-MS/MS. Total Tau protein (t-Tau) and phosphorylated Tau protein (p-Tau), markers of AD, were assessed by Western blot. The mean mass concentration of PM2.5 was 8.6 μg/m3 from the first 3 months exposure and 10.8 μg/m3 from the second 3 months exposure. Our results showed that MWM didn’t have any differences compared to control groups after exposure for 3 months. Both control and exposure groups showed a reasonable learning curve of escape latency in acquisition phase. In MDA, the level of MDA significantly increased in olfactory bulb, hippocampus and cerebral cortex after 6 months exposure. In 3 months exposure, the MDA level in four brain regions didn’t find any significant differences between control and exposure group. The result of Tau protein expression showed that the level of t-Tau protein expression, as well as p-Tau protein expression, significantly increased in olfactory bulb in 3 months exposure. However, in 6 months exposure, the level of t-Tau and p-Tau protein expression didn’t find any significant differences between control and exposure group in four brain regions. We found no significant differences between the control and the exposure group in histopathology of lung from 3 and 6 months exposure and brain from 6 months exposure. In conclusion, our study indicated the subchronic exposure to ambient particles would induce oxidative stress and Tau protein in brain. However, exposure to ambient particles for 3 months may not impair spatial learning and memory in SHR. Our study revealed the possible mechanism of PM exposure by inhalation and neurotoxicity in CNS. Nevertheless, our study didn’t investigate many neurotoxicity-related markers in CNS. Further study should be conducted to explore the more markers related to neurotoxicity in CNS and clarify the relationship between PM and neurotoxicity in CNS. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70620 |
DOI: | 10.6342/NTU201802983 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 職業醫學與工業衛生研究所 |
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