臺灣博碩士論文加值系統

帕金森氏症 (Parkinson’s disease) 是很常見的神經退化性疾病，僅次於阿茲海默症，目前僅能依靠藥物減緩病症的發生，但無法完全根除，而發病的主要症狀為靜止性震顫，運動遲緩和肌肉僵直。帕金森氏症的發病機轉，是因為大腦黑質緻密部的多巴胺神經元流失所導致，α-synuclein 的基因突變則是造成流失的原因，主要有三種類型的突變，包含 A53T，A30P 和 E46K，在發病的機制中有著重大的影響。在此實驗我們想利用 A53T 轉基因小鼠來模擬病人利用自身的誘導型多功能幹細胞進行基因治療後來降低突變基因的表現，並將幹細胞分化成神經幹細胞並進行移植，進而讓移植的神經幹細胞取代原本腦內受損的神經元細胞，達到治療的效果。在帕金森氏症小鼠模型中，我們利用 Rotarod、Beamwalking 與Locomotor 評估移植與否的差別，持續追蹤至十個月大，也利用免疫螢光染色證實移植基因治療後的神經幹細胞小鼠，在黑質部位的多巴胺神經元數量有顯著上升，這些數據都證實治療組比控制組來的有顯著效果，而處理過基因治療組比起沒有基因處理過的組別發病時間明顯晚了許多。我們的研究顯示降低α-synuclein表現的 iPS 細胞分化的神經幹細胞可治療A53T 缺陷的帕金森氏症患者。這對未來臨床治療的發展將大有幫助。

Parkinson’s disease (PD) is the most common neurodegenerative disease and it has no effective therapeutic method. The symptoms of PD include resting tremor, bradykinesia and muscle rigidity. PD pathogenesis is characterized by loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). α-synuclein (SNCA) which has three types of missense mutations, A53T, A30P, and E46K, which play an important role in pathogenesis of Parkinson’s disease. The SNCA-A53T mutation transgenic mice model (PD mouse model from The Jackson Lab) developed PD around 8 months of age. Stem cells provide hope for PD. In all type of stem cells, cells (including iPS cells) are the most powerful cells that could differentiate into all-three kinds of germ layers, including DA neurons.
In our experiment, we will create iPS-SNCA from SNCA-A53T mutation transgenic mouse fibroblast. The iPS-SNCA cells will silence the SNCA-A53T mutation gene by using shRNA system to achieve therapeutic effect and differentiate into neural stem cell (NSC) to treat SNCA-A53T mice to confirm that the NSC is useful therapeutically. Finally, the behavior test data will used to evaluate the therapy efficiency of the NSC transplanted by locomotor activity, beam walking and rotarod.
In summary, we demonstrated that the IPS is a powerful technology to rescue Parkinson patients.

目錄
致謝 I
圖目錄 V
縮寫對照表 VI
中文摘要 VII
英文摘要 VIII
壹、 緒論 1
1. 帕金森氏症（Parkinson’s Disease） 1
2. 多巴胺 2
3. alpha-synuclein (SNCA) 的功能 2
4. 誘導型多功能幹細胞 (induced pluripotent stem cells, iPS) 3
5. iPS-SNCA 細胞 5
6. 神經幹細胞 (Neural stem cells, NSC) 5
7. 1-methyl-4-phenyl--phenylpyridinium (MPP+) 6
貳、 研究架構與動機 7
叄、材料與方法 8
1 實驗材料 8
1.1 材料 8
1.2 試劑 8
1.3 實驗儀器與耗材 12
2 實驗方法 13
2.1 轉基因小鼠胚胎纖維母細胞取得與培養 13
2.2 建立A53T 轉基因之iPS細胞 14
2.3 鹼性磷酸酶染色 (Alkaline phosphatase staining, AP) 14
2.4 免疫螢光染色(Immunofluorescence staining) 15
2.5 幹細胞形成胚球體與自發性分化 16
2.6 誘導iPS細胞分化為神經幹細胞 16
2.7 測試shRNA 降低 SNCA的效果 18
2.8 Lentivirus病毒製作與感染 18
2.9 RNA 萃取與cDNA 製備 19
2.10 即時聚合酶連鎖反應 (Real-time PCR) 20
2.11 西方墨點法 20
2.12 神經幹細胞移植 22
2.13 動物行為模式分析 22
2.14 冷凍切片 23
2.15 組織免疫螢光染色分析 24
2.16 TUNEL assay 25
肆、結果 26
4.1 確認纖維母細胞的突變序列 26
4.2 建立 iPS-SNCA 細胞 26
4.3 iPS-SNCA 於細胞體外環境下分化為神經幹細胞 27
4.4 使用shRNA 系統降低 SNCA 於神經幹細胞內的表現量 27
4.5 利用行為模式評估帕金森氏症小鼠損傷程度以及修復程度 28
4.6 免疫螢光染色觀察 iPS-SNCA 分化之神經幹細胞在 A53T 小鼠腦中的修復程度 29
4.7 使用TUNEL assay 探討細胞移植的效果 30
伍、討論 31
陸、參考文獻 32
柒、圖表 37
捌、附圖 48

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