第三型小腦萎縮症為一種遺傳性神經退化疾病，其致病主因為突變ATXN3基因上CAG序列重複次數較正常人高，導致突變ATXN3蛋白上帶有過長的聚麩醯胺酸長鏈。突變 ATXN3蛋白會經過蛋白質水解酵素 (如鈣蛋白酶)剪切後產生小分子量蛋白水解片段，進一步形成非水溶性ATXN3蛋白聚集體累積於肯浦野神經細胞並使其凋亡。TSCA-001是一個小分子藥物，在先前研究中於基因轉殖之斑馬魚與小鼠動物模型中已驗證其具有延緩肯浦野神經退化的功效。由於病患衍生之誘導型多能幹細胞帶有疾病患者的致病基因，亦有與胚胎幹細胞相近的分化能力，因此本研究以第三型小腦萎縮症病患衍生之誘導型多能幹細胞作為藥物有效性與機制探討的平台。以流式細胞儀搭配專一性抗體驗證，發現第三型小腦萎縮症病患衍生之誘導型多能幹細胞，能夠成功分化為肯浦野前驅細胞。進一步使用喹啉酸增加此細胞模型內鈣蛋白酶的活性後，可誘發典型第三型小腦萎縮症之症狀，包含非水溶性 ATXN3蛋白聚集體的生成與細胞凋亡現象。本研究也發現TSCA-001之處理能抑制經喹啉酸刺激所活化之鈣蛋白酶，並減少原本堆積於細胞核之非水溶性ATXN3蛋白聚集體。因此，TSCA-001抑制了因喹啉酸導致的細胞凋亡現象。綜上結果，本研究應用病患衍生之誘導型多能幹細胞驗證了TSCA-001對於第三型小腦萎縮症之療效，並提出其治療機轉。

　　Spinocerebellar ataxia type 3 (SCA3) is a kind of dominantly inherited neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) encoding CAG trinucleotide repeats in ATXN3. The mutant ataxin 3 (ATXN3) is cleaved by proteolytic enzymes, such as by calpain, and leads to the formation of intranuclear aggregates which eventually result in atrophy of Purkinje cells in spinocerebellum. TSCA-001, a small molecule drug, has previously been demonstrated to delay the disease progressions of SCA3 by preventing the atrophies of Purkinje cells in transgenic mouse and zebrafish models. Based on the patient-specific gene expression and differentiation potentials, patient-derived induced pluripotent stem cells (iPSCs) were used as a useful platform to study the drug efficacy and mechanisms for SCA3 in this study. It was shown that Purkinje progenitor cells could be successfully differentiated from induced pluripotent stem cells (iPSCs) derived from SCA3 patients by analyzing the cell-specific markers with immunofluorescence and flow cytometry. Furthermore, it was found that, in the Purkinje progenitor cells differentiated from SCA3 patient-derived iPSCs, the exposure to quinolinic acid triggered intranuclear ataxin 3 aggregation and neuronal death, which were typically observed in the spinocerebellum of SCA3 patients. It was also found that the treatments with TSCA-001 could significantly decrease calpain activities triggered by quinolinic acid and reduce insoluble ataxin 3 aggregation in the nucleus. The quinolinic acid-induced apoptosis in Purkinje progenitor cells differentiated from SCA3 patient-derived iPSCs could therefore be ameliorated. A putative underlying mechanism was proposed for the therapeutic effects of TSCA-001 on SCA3 pathogenesis.

目錄：
中文摘要 I
英文摘要 III
目錄 V
圖目錄 IX
表目錄 XIII
一、研究動機與研究目的-1
二、研究背景介紹-3
2.1 第三型小腦萎縮症簡介-3
2.1.1 第三型小腦萎縮症 (Spinocerebellar ataxia type 3, SCA3)-3
2.1.2 突變ATXN3致病機制-3
2.2 誘導型多能幹細胞 (Induced pluripotent stem cells, iPSCs)-5
2.3 肯浦野細胞 (Purkinje cells)-7
2.4 TSCA-001 -8
2.5 肯浦野前驅細胞的分化-8
2.6 鈣蛋白酶的過度活化對肯浦野前驅細胞的影響-9
三、材料與方法-11
3.1 實驗設計-11
3.2 實驗方法-12
3.2.1 誘導型多能幹細胞的繼代與培養-12
3.2.2 誘導型多能幹細胞的幹細胞特性鑑定-12
3.2.3 肯浦野前驅細胞分化-13
3.2.4 SCA3組肯浦野前驅細胞加速疾病進程與TSCA-001加入處理-14
3.2.5 西方點墨法 (Western blot)-15
3.2.6 免疫螢光染色-18
3.2.7 流式細胞儀測定分化後細胞的比率-19
3.2.8 神經細胞相關基因表現-20
3.2.9 鈣蛋白酶活性測定-21
四、實驗結果-23
4.1 Normal與SCA3誘導型多能幹細胞的鑑定和ATXN3蛋白之比較-23
4.1.1 誘導型多能幹細胞的相關表徵分析-23
4.1.2 Normal與SCA3的誘導型多能幹細胞的ATXN3蛋白質差異-24
4.2 肯浦野前驅細胞的分化與分析鑑定-25
4.2.1分化後細胞的類神經結構表現-25
4.2.2 分化後的細胞可表現神經細胞與肯浦野前驅細胞相關蛋白-27
4.2.3 分化後的細胞可表現神經細胞與肯浦野前驅細胞的指標基因-28
4.2.4 分化後的細胞比例-29
4.3加速SCA3組肯浦野前驅細胞的疾病進程與TSCA-001的影響-30
4.3.1 喹啉酸與麩胺酸使SCA3組肯浦野前驅細胞產生類神經結構萎縮-31
4.3.2 喹啉酸方法與麩胺酸方法加速疾病進程後所得非水溶性ATXN3蛋白質聚集體之差異-34
4.3.3加速疾病進程後的SCA3組肯浦野前驅細胞ATXN3蛋白多分布於核內-37
4.3.4 TSCA-001減少了加速疾病進程後的SCA3組肯浦野前驅細胞的非水溶性ATXN3蛋白聚集體-39
4.3.5 TSCA-001降低加速疾病進程後的SCA3組肯浦野前驅細胞凋亡現象與鈣蛋白酶活性-41
五、討論-45
六、結論-51
七、參考文獻-53

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