臺灣博碩士論文加值系統

摘要
第3型脊髓性小腦萎縮症為一種神經退化性疾病，致病原因為ATXN3基因發生異常的CAG序列重複，導致Ataxin-3(ATXN3)蛋白聚集，其疾病特徵為小腦神經元逐漸死亡且運動功能逐漸喪失。目前臨床上所利用之藥物或物理治療方式，皆無法有效延長病人壽命及提升其生活品質。TSCA-001乃由中草藥萃取之小分子藥物，本研究使用細胞模型、斑馬魚疾病模式及轉殖基因小鼠，探討TSCA-001治療神經退化性疾病第3型脊髓性小腦萎縮症之可行性及其作用機制。 本研究在細胞模式中發現TSCA-001可顯著減少因ATXN3蛋白不正常重複聚麩醯胺酸(polyQ)所產生之聚集現象。進一步在剔除ATXN3 斑馬魚(Huc-GFP)疾病模式中發現，TSCA-001可顯著維持斑馬魚運動神經細胞之數量，並在斑馬魚觸碰實驗中發現其游動狀態有顯著回復。另外，以MJD84.2(ATXN3*)轉殖基因小鼠疾病模式進行TSCA-001口服療程，結果顯示TSCA-001可顯著改善其四肢運動能力、平衡能力及抓握能力;在組織化學免疫染色及西方墨點法分析，發現 TSCA-001可顯著維持浦肯野細胞之數目及增加泛素進行泛素化能力。綜合以上結果，TSCA-001可維持運動神經元之數目並減緩運動能力喪失且可降低突變ATXN3之聚集在第3型脊髓性小腦萎縮症中，故TSCA-001 未來有成為治療神經退化性疾病之臨床新藥的潛力。

Abstract
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease caused by an expansion of CAG sequence repeats on the ATXN3 gene and further leading to the aggregation of abnormal ataxin-3 (ATXN3) protein. The disease can be characterized by the degeneration of cerebellar neurons and the loss of motor function. SCA3 is an incurable disease, because the present treatment strategy including drugs and physical therapy can neither effectively prolong the life span nor improve the quality of life of the patients. For the above reasons, there is a strong unmet medical need for an effective therapeutic strategy. TSCA-001 is a novel small molecule drug originally extracted from herbal source. In this study, both in vitro and in vivo experiments were performed to confirm TSCA-001’s therapeutic potential and possible underlying mechanism for SCA3 treatment. The results showed that abnormal ATXN3 protein’s aggregation decreased in the cells treated with TSCA-001. It was also observed that TSCA-001 could significantly maintain the number of motor neurons and recover its motor function in the ATXN3 knockout zebrafish model. Furthermore, our results indicated that the MJD84.2 (ATXN3*) SCA3 mice model treated with TSCA-001 for 3.5 months revealed a significant improvement of the motor function. Maintenance of the Purkinje cells in the cerebellum was found in the TSCA-001 treated SCA3 mice. Western blotting data also confirmed this result and further revealed that the ubiquitination ability in the cerebellum was enhanced by TSCA-001. In conclusion, treatment of TSCA-001 can delay the disease progress of SCA3, possibility due to the decreased the aggregation of mutant ATXN3 protein, thereby allowing an improved motor function in SCA3 mice. Therefore TSCA-001 demonstrated a great potential in the treatment of Spinocerebellar ataxia type 3.

目錄
摘要 I
Abstract III
目錄 V
圖目錄 VIII
表目錄 IX
一、研究動機與研究目的 1
二、背景及重要性簡介 2
2.1.脊髓性小腦萎縮症 2
2.2脊髓性小腦萎縮症第三型(Spinocerebellar ataxia type3, SCA3) 5
2.3 Ataxin-3(ATXN3)蛋白 6
2.4斑馬魚動物模型 7
2.5 MJD84.2(ATXN3*)轉殖基因小鼠動物模型 8
三、研究材料與方法 10
3.1實驗設計 10
3.1.1.實驗流程圖 10
3.2實驗方法 11
3.2.1 HEK293細胞繼代培養 11
3.2.2 Plasmid 11
3.2.3細胞轉染 13
3.2.4西方點墨法 (Western Blotting) 13
3.3 動物實驗 13
3.3.1斑馬魚品系 13
3.3.2 Morpholino DNA片段 13
3.3.3 螢光顯微鏡觀察 14
3.3.4 行為測試 14
四、結果 16
4.1 TSCA-001對於斑馬魚存活率之影響 16
4.2 TSCA-001對疾病斑馬魚行為影響 17
4.3 TSCA-001作用於神經損傷斑馬魚 18
4.4細胞實驗 19
4.5 TSCA-001改善SCA3小鼠行為模式 21
4.6 TSCA-001 對神經細胞影響 24
4.7 Western Blot組織蛋白分析 26
五、討論 28
六、結論 31
七、參考文獻 33

圖目錄
圖一、全世界SCA各型分布及比例圖 4
圖二、ATXN3作用圖 7
圖三、Plasmid結構圖 12
圖四、TSCA-001對斑馬魚存活率之影響 16
圖五、TSCA-001對斑馬魚行為之影響 17
圖六、TSCA-001對於神經損傷斑馬魚之作用 18
圖七、轉染細胞加入TSCA-001蛋白螢光比較 20
圖八、SCA3小鼠之平衡能力及抓握能力 21
圖九、TSCA-001對於SCA3小鼠行為之影響 23
圖十、SCA3小鼠犧牲免疫化學組織染色小腦切片 25
圖十一、SCA3小鼠犧牲免疫化學組織染色小腦切片統計圖 25
圖十二、Western Blot 檢測 SCA3小鼠小腦ubiquitination蛋白 26
圖十三、Western Blot 檢測SCA3小鼠小腦Calbindin蛋白 27
圖十四、TSCA-001對斑馬魚與小鼠之作用簡圖及機制假說圖 32


表目錄
表一、polyQ病理特性 4
表二、台灣SCA各型比例 5

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