臺灣博碩士論文加值系統

神經發炎在於神經病變疾病中的病因學和病情發展扮演著重要角色，例如，阿茲海默症。腦源性神經滋養因子(Brain-derived neurotrophic factor, BDNF)為中樞神經系統中分布最廣泛之滋養因子，對於神經元細胞存活與突觸的可塑性扮演著重要的角色。此研究目的是探討利用低強度脈衝超音波(low-intensity pulsed ultrasound, LIPUS)對脂多醣(Lipopolysaccharide, LPS)誘導的神經發炎以及記憶損傷模擬的阿茲海默症模型中的神經保護效果。
在細胞實驗中，我們利用LIPUS刺激星狀膠細胞，星狀膠細胞增加細胞的存活率並且增加了BDNF的表現量；而且，LIPUS可以緩解因Aβ(amyloid β)刺激後引起的星狀膠細胞的死亡。在動物實驗中，在水迷宮實驗結果顯示給予LPS造成神經發炎的小鼠停留在目標區域的時間小於健康小鼠，而使用LIPUS治療後顯著增加其停留在目標區域的滯留時間。此外；在物件辨識的行為實驗中，LIPUS治療組別的表現亦優於未治療的LPS組。此外，蛋白質分析的結果顯示，經LIPUS治療後能顯著減緩海馬迴及皮質的區域中的發炎因子(TNF-α，IL-1β和IL-6)，可知LIPUS具有減緩神經發炎的功能。
綜合以上結果，我們發現LIPUS能夠減緩因脂多醣所造成的記憶缺損並且透過抑制神經發炎與BDNF的減損，並且可減少類澱粉蛋白的沉積。因此，LIPUS刺激對於如阿茲海默症等之神經退化性疾病提供一項具有潛力的治療方法。

Neuroinflammation has been known to play a critical role in the etiology and progression of several neurodegenerative disorders, such as Alzheimer’s disease (AD). Brain-derived neurotrophic factor (BDNF), the most prevalent neurotrophin in the central nervous system (CNS), plays a key role in neuronal survival and synaptic plasticity. The aim of this study was to investigate the protective role of low-intensity pulsed ultrasound (LIPUS) against lipopolysaccharide (LPS)-induced neuroinflammation and memory impairments in a simulation to AD.
In in vitro experiment, we used LIPUS to stimulate the astrocytes. LIPUS increased the cell viability and BDNF expression. Moreover, the death of astrocytes induced by Aβ can be alleviated following LIPUS stimulation. In in vivo experiment, our data illustrates that mice in LPS group spent less time in the target quadrant than the sham group. LPS plus LIPUS-treated mice exhibited a significant increase in the average time spent in the target quadrant compared to the LPS-treated group. Furthermore, LPS plus LIPUS-treated mice revealed a preference for the novel object compared to the LPS-treated group. In addition, the levels of proinflammatory cytokines (TNF-, IL-1β, and IL-6) in the hippocampus or cortex regions of LPS-treated mice were significantly decreased following LIPUS treatment, suggesting the finding that LIPUS is a promising tool which has the property to attenuate neuroinflammation.
In summary, our results showed that LIPUS attenuated LPS-induced memory impairment as well as amyloidogenesis via suppression of neuroinflammatory activity and BDNF decline. Thus, LIPUS stimulation might be a useful intervention for neuroinflammation-associated Alzheimer’s disease.

致謝 I
中文摘要 II
ABSTRACT IV
目錄 VI
圖目錄 VIII
表目錄 IX
縮寫表 X
第一章緒論 1
1.1 神經發炎反應 1
1.2 脂多醣(LPS) 2
1.3 阿茲海默症 3
1.4 星狀膠質細胞 4
1.5 腦源性神經滋養因子 6
1.6 超音波的運用 7
1.7 研究動機與目的 9
第二章 實驗材料與方法 10
2.1 實驗藥品來源 10
2.2 細胞實驗 12
2.2.1 實驗細胞來源 12
2.2.2 實驗溶液配製 12
2.2.3 細胞解凍 13
2.2.4 細胞培養 14
2.2.5 繼代培養 14
2.2.6 冷凍細胞 14
2.2.7 細胞實驗流程 15
2.2.8 細胞實驗分組 16
2.3 細胞增殖分析 16
2.4 動物實驗 17
2.4.1 實驗動物來源 17
2.4.2 動物實驗流程 17
2.4.3 動物實驗與分組 18
2.5 動物行為實驗 19
2.5.1 水迷宮實驗 19
2.5.2 物件辨識 20
2.6 超音波實驗設備 20
2.6.1 平面脈衝式超音波設備 20
2.6.2 聚焦脈衝式超音波設備 21
2.6.3 超音波參數設定 22
2.7 蛋白質萃取 23
2.8 西方墨點法(Wetern Bolt) 24
2.8.1 實驗溶液配置 24
2.8.2 蛋白質定量 24
2.8.3 電泳膠製備與分析 25
2.8.4 轉印 26
2.8.5 目標物與抗體結合 26
2.9 統計方法 28
第三章 實驗結果 29
3.1 脈衝式超音波增加細胞內神經滋養因子表現量 29
3.2 A對於細胞的影響 31
3.3 脈衝式超音波減少A對細胞產生的毒殺性 33
3.4 脈衝式超音波對老鼠體重變化的影響 35
3.5 水迷宮測試學習及記憶能力 36
3.6 物件辨識行為測試認知能力 41
3.7 脈衝式超音波對類澱粉蛋白沉積的影響 42
3.8 脈衝式超音波對腦內神經膠質細胞的影響 46
3.9 脈衝式超音波影響腦內神經發炎因子 49
3.10 脈衝式超音波影響腦內神經發炎反應 52
3.11 脈衝式超音波影響動物神經滋養因子表現量 55
3.12 脈衝式超音波對BDNF晚期的影響 58
第四章 討論 60
4.1 脈衝式超音波降低A細胞毒性 60
4.2 脈衝式超音波改善動物行為模式 61
4.3 脈衝式超音波對於BDNF的刺激 63
4.4 神經膠質細胞的探討 65
4.5 脈衝式超音波對於發炎因子的影響 67
第五章 結論與未來展望 68
文獻參考 70

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