臺灣博碩士論文加值系統

第十七型脊髓小腦運動失調症 (SCA17) 是一種體染色體顯性遺傳神經退化性疾病，患者有漸進式的小腦萎縮、共濟失調、癲癇及認知障礙等病徵。然而，目前沒有任何具有顯著療效的藥物可以治療SCA17。本研究旨在探討高壓氧治療(HBOT)對SCA17 TG小鼠行為改善及神經保護的作用。HBOT是一種無痛、較為非侵入性的治療方式，患者於充滿100%氧氣且高壓的艙室中進行治療；目前已知可用於治療一氧化碳中毒、動脈栓塞、創傷性腦損傷、燒燙傷及減壓病(潛水夫病)等；目前已有文獻證實，HBOT可以降低發炎反應的發生、改善組織缺氧的狀態，並促使微血管增生。本研究採用SCA17基因轉殖小鼠，進行各式行為實驗(曠野實驗、Ｙ字迷宮、步態分析及滾輪測試)評估其運動和空間記憶的能力；經過HBOT，我們發現SCA17 TG小鼠的焦慮情況、短期記憶及運動協調能力有顯著地改善。我們也以每個月進行一次行為實驗之方式得知HBOT效果的持續性可以維持三個月。而輕微高壓氧(M-HBOT)能夠使小鼠短期記憶及運動協調能力有顯著地改善。在小腦組織病理切片結果中，我們發現，HBOT降低了SCA17 TG小鼠小腦中Purkinje cell丟失，也使其排列順序變整齊、細胞本體較為飽滿及神經纖維密度上升；也發現HBOT能夠抑制SCA17 TG小鼠減緩其神經發炎反應astrocyte及microglia的發生；另外，經HBOT之SCA17 TG小鼠與記憶相關及細胞增殖之蛋白路徑AKT/CaMKII -ERK-CREB在小腦中磷酸化之蛋白表現量有顯著的上升。因此，我們認為研究結果說明HBOT對於SCA17具有治療的發展潛力。

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant inherited neurodegenerative disease. Patients show progressive cerebellar atrophy, ataxia, epilepsy and dementia. However, no drug with significant effect on the treatment of SCA17 has been developed. This study was designed to investigate the effects of hyperbaric oxygen therapy (HBOT) on neuroprotection in SCA17 TG mice. HBOT is a painless, less-invasive treatment that is widely used for carbon monoxide poisoning, arterial embolism, traumatic ischemia, burns and decompression sickness. Patients are treated in a cabinet with high atmospheric pressure and 100% oxygen for a period of time. It is reported HBOT reduces the inflammatory re-sponse and hypoxia in tissue, and improves microvascular proliferation. In this study, behavior tests on SCA17 transgenic mice were performed before and after HBOT, including the open-field, Y-maze, footprint and rotarod. The results showed that HBOT had no effect in the improvement of hyperactivity and gait ataxia according to the open-field and footprint tests, while the results from open-field, Y-maze and rotarod tests showed HBOT significantly improved the anxiety, short-term memory and motor-coordination of SCA17 mice. After the monthly behavioral evaluation, we learned that the duration of this HBOT effect can be lasted for three months. However, mild hyperbaric oxygen (M-HBOT) can only significantly improve short-term memory and motor coordination in SCA17 mice. Therefore, we believed that HBOT has therapeutic potential for SCA17. In the pathological examination of cerebellum, we found that HBOT reduced the loss of Purkinje cells in the cerebellum of SCA17 TG mice, made the cells in a more order pattern, enlarged the cell body , and the increased the nerve fiber density. It was also found that HBOT ameliorated the neuroinflammatory response of SCA17 mice. In addition, the HBOT increased the proteins associated with memory pathway, AKT/CaMKII, ERK, and CREB in the mouse cerebellum. These findings suggest that HBOT has therapeutic potential for SCA17.

目錄
1. 中文摘要 1
2. Abstract 3
3. 縮寫表 5
4. 研究背景 9
4.1 多麩醯胺酸擴增疾病（Polyglutamine expansion diseases） 9
4.2 脊髓小腦運動共濟失調症（Spinocerebellar ataxias） 10
4.3 第十七型脊髓小腦共濟失調症（SCA17） 11
4.4 Purkinje cell (PC) 12
4.5 神經退化性疾病與氧化壓力(Neurodegenerative diseases with oxidative stress) 13
4.6 神經發炎(Neuroinflammation) 14
4.7 SCA17基因轉殖小鼠(SCA17 transgenic mice, SCA17 TG mice) 15
4.8 高壓氧治療 (Hyperbaric oxygen treatment, HBOT) 15
5. 研究目的研究動機與目的 17
6. 材料與方法 18
6.1 基因轉殖小鼠之產生及飼育 18
6.2 基因轉殖鼠之基因型分析 (Genotyping) 18
6.3 高壓氧治療 (Hyperbaric oxygen therapy, HBOT) 20
6.4 高壓氧治療效果持續性實驗設計 20
6.5 高壓氧治療效果病理與行為分析之實驗設計 21
6.6 小鼠血液相關分析 21
6.7 氧化壓力測定 22
6.8 輕微高壓氧治療(Mild-HBOT)效果實驗設計 24
6.9 西方墨點法 (Western blotting) 25
6.10 小腦冷凍切片 26
6.11 免疫螢光染色 (Immunofluorescence staining, IF) 26
6.12 免疫組織化學染色 (Immunohistochemistry, IHC) 27
6.13 動物行為分析 28
曠野試驗(Open field) 28
Y字迷宮(Y-maze) 29
步態分析(Footprint) 30
滾輪測試(Rota-rod task) 30
6.14 統計分析(Statistical analysis) 31
7. 研究結果 32
7.1 HBOT對小鼠行為之影響與治療持續性 32
7.2 HBOT對TG-HBO小鼠小腦中calbindin與GFAP蛋白表現量之影響 33
7.3 HBOT對小鼠氧化壓力之影響 34
7.4 HBOT對促進小鼠小腦神經細胞增殖及改善認知障礙相關蛋白表現之影響 35
7.5 HBOT對分子伴侶介導的自噬作用 (Chaperone-mediated autophagy, CMA) 相關蛋白HSC70表現之影響 36
7.6 HBOT對小鼠小腦中樹突生長相關蛋白GAD67表現程度之影響 36
7.7 以小腦組織切片免疫螢光染色評估HBOT對小鼠小腦蛋白表現之影響 37
7.8 以小腦組織切片IHC染色評估HBOT對小鼠小腦microglia之影響 39
7.9 以小量動物實驗評估輕微高壓氧治療 (Mild HBOT, M-HBOT) 對SCA17 TG小鼠之效果 39
7.10 血液檢查評估HBOT對小鼠基礎健康狀況之影響 40
8. 討論 42
9. 參考資料 50
10. 圖表 62
圖1、HBOT對小鼠行為之影響與治療持續性 62
圖2、分析HBOT對SCA17 TG小鼠小腦中calbindin與GFAP蛋白表現量之影響 66
圖3、分析HBOT對小鼠氧化壓力之影響 68
圖4、分析HBOT對促進小鼠小腦神經細胞增殖及改善認知障礙相關蛋白表現之影響 70
圖5、分析HBOT對分子伴侶介導的自噬作用 (Chaperone-mediated autophagy, CMA) 相關蛋白HSC70表現之影響 76
圖6、分析HBOT對小鼠小腦中樹突生長相關蛋白GAD67表現程度之影響 77
圖7、以小腦組織切片免疫螢光染色評估HBOT對小鼠小腦蛋白表現之影響 78
圖8、小腦組織切片進行IHC染色評估HBOT對小鼠小腦microglia之影響及定量結果 86
圖9、評估M-HBOT對小鼠之神經保護效果 88
圖10、以血液檢查評估HBOT對小鼠之影響 90
圖11、HBOT可能活化細胞增殖及神經保護之分子機制相關路徑 94
表一、抗氧化檢測試劑組Trolox標準品配置方法 95
表二、初級抗體列表 96
表三、次級抗體列表 97
表四、行為實驗總表 97
表五、公鼠蛋白質分析總表 98
表六、母鼠蛋白質分析總表 99
表七、公鼠腦片免疫螢光染色及免疫化學染色定量總表 100
表八、母鼠腦片免疫螢光染色及免疫化學染色定量總表 101
11、補充資料 102
11.1 國家實驗動物中心肝腎病理分析報告 102

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