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研究生： 邱乃華
Chiu, Nai-Hua
論文名稱： 標靶聲遺傳學結合機電蛋白應用於帕金森氏症治療
Targeted Sonogenetics for Parkinson's Disease Treatment with Electromechanical Protein
指導教授： 葉秩光
Yeh, Chih-Kuang
口試委員: 林玉俊
Lin, Yu-Chun
林靜嫻
Lin, Chin-Hsien
學位類別： 碩士
Master
系所名稱： 原子科學院 - 核子工程與科學研究所
Nuclear Engineering and Science
論文出版年： 2019
畢業學年度： 108
語文別： 中文
論文頁數： 73
中文關鍵詞： 超音波 、機械敏感離子通道 、神經退化性疾病 、帕金森氏症 、基因遞送
外文關鍵詞： ultrasound, mechanosensitive ion channel, neurodegeneration, Parkinson’s disease, gene delivery
相關次數： 點閱：3  下載：0 
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    • 帕金森病為目前第二大宗之神經退行性疾病，肇因於腦部主要控制運動區域-黑質緻密部的多巴胺神經元逐漸退化，使神經傳導物質多巴胺神經元日益分泌不足，最終影響腦部控制肌肉運動，產生動作障礙。儘管抗神經退化療法已在多種動物模型中取得正面效果，但仍有缺點，例如：藥物療法可能具有藥物毒性，並且隨著帕金森氏症的惡化，左旋多巴胺的需求量也會上升，最後容易造成多巴胺失調症與藥物依賴性。深層腦部電刺激屬於侵入性手術，具有腦損傷或是產生併發症的可能。基因治療則是仍有潛在免疫反應、無法精準控制基因轉殖區域等問題，另外遞送基因載體之方法大多是屬於侵入式的穿顱注射。因此，目前尚無方法可達成非侵入式、局部性之抗神經退化或神經保護治療。
    我們的研究團隊先期已建立出聲遺傳學系統，此系統由超音波與對超音波敏感之prestin蛋白組成，可達成非侵入式刺激特定細胞的目的。Prestin蛋白為一種機電蛋白，存在於迴聲定位動物的耳蝸之外毛細胞，許多研究均指出此蛋白對於高頻聽覺至關重要。本研究之目的為將工程改造之prestin蛋白轉殖至帕金森氏動物之多巴胺神經元，再透過超音波調控多巴胺神經元的活性與腦中神經滋養因子的分泌，使已退化之多巴胺神經重新修復甚至再生，最終改善帕金森氏症之症狀。
    本研究提供兩個策略進行活體腦部基因轉殖：第一個策略是使用微氣泡作為基因載體，可藉由超音波驅動，同時開啟血腦屏障並遞送基因，進行非侵入式的活體基因轉殖。細胞實驗結果顯示最佳聲學轉殖參數是聲壓為0.5 MPa、週波數為5000、脈衝重複頻率為10 Hz，可達到15.47 ± 3.04 %的轉殖率；動物實驗顯示最佳聲學轉殖參數是聲壓為0.5 MPa、照射時間為240 秒、轉殖後48小時表現量最大，且腦部無出血性傷害，但整體來說轉殖率偏低且無法精準控制將基因僅遞送到多巴胺神經元區域。
    第二個策略是使用腺病毒作為基因載體，將病毒穿顱注射至黑質區域。實驗發現Prestin蛋白在轉殖後一星期開始表現，至少可維持八個星期； c-fos染色與多巴胺神經細胞具有70 %的重合率，證實表達Prestin蛋白的多巴胺神經可被超音波刺激；此外，治療後的老鼠可以分別提升 3.8 倍的平衡能力及 1.4 倍的運動能力，在免疫染色分析與西方墨點分析法可以觀察到治療老鼠分別可恢復73%之TH+表現量與提升8倍的TH+含量，表示治療過後，動物之多巴胺神經元確實已被修復，並改善帕金森氏症鼠的運動能力。
    綜合以上資料，本研究成功提出一項利用超音波達成非侵入式調控腦部特定細胞之活性的技術，可克服傳統方法面臨之使用限制。未來工作將探討此技術之活體安全性並嘗試應用於不同神經退化疾病治療。

    Parkinson’s disease (PD) is a neurological disorder that involves a profound loss and dysfunction of dopaminergic neurons in substantia nigra (SN), resulting in dopamine depletion, motor and other non-motor symptoms. Recently, interrupting the neurodegenerative processes of PD has arisen a potential treatment option because it can slow or stop the disease progression. However, conventional strategies (electrical stimulation, pharmacotherapy or, gene therapy) frequently produce various complications. In other words, all existing methods remain symptomatic, and no therapy is currently available to achieve non-invasively and locally promote neural regeneration and neuroprotection.
    We recently established a sonogenetic technology which was consisted of ultrasound (US) and an US-sensitive protein, prestin for non-invasively activating specific cell. The prestin is an electromechanical protein, which resides in the outer hair cells of echolocating mammals’ cochlea and is important for high-frequency hearing. Here we expressed the engineered prestin in dopaminergic neurons at SN of PD mice and showed dopaminergic neurons activity and neurotrophin expression could be remotely modulated by US, relieving PD symptoms.
    Two kinds of gene delivery methods were used to transfect prestin into mouse brain. First, the prestin was delivered by utilizing non-invasive and non-viral ultrasonic disruption of the blood-brain barrier via Prestin-loaded microbubbles (Prestin-MBs). The maximum Prestin expression with the highest cell viability occurred at a pressure of 0.5 MPa, cycle number of 5000, and PRF of 1 Hz. In vivo data suggest that the optimal parameters for expressing a transgene are 0.5 MPa with a sonication time of 240 s, 48 h later, in an US-targeted region, while minimizing erythrocyte extravasation. However, the in vivo gene expression rate is still too low to achieve sufficient stimulation.
    Second, the prestin was delivered by transcranial injecting viral vector into SN area. The prestin expression was detected in dopaminergic neurons at 1 week after injection and remained detectable at least 8 weeks, suggesting the period of prestin expression was enough for US treatment. The highly co-localization between c-fos positive and TH positive signals (70 %) confirmed the successful activation of dopaminergic neurons. Additionally, US stimulation resulted in 3.8 and 1.4 folds of balance ability and motor ability improvement compared to untreated PD mice, individually.
    In conclusion, this technology allows a noninvasive procedure to specific brain regions capable of being selectively modulated using US, overcoming the key limitations of conventional brain therapies. Future works include long-term assessing the safety issue and apply into different neurodegenerative diseases.

    摘要 i Abstract iii 目錄 v 表目錄 viii 圖目錄 ix 1 緒論 1 1.1 帕金森氏症之治療 1 1.1.1 帕金森氏症(Parkinson’s Disease) 1 1.1.2 藥物治療 2 1.1.3 深層腦部電刺激 3 1.1.4 基因治療 3 1.1.5 目前帕金森氏症療法之治療困境 4 1.2 光基因遺傳治療 5 1.3 超音波誘發細胞內外鈣離子流通 6 1.3.1 超音波 6 1.3.2 使用聲遺傳學提升神經細胞對超音波之敏感度 8 1.4 哺乳動物聽覺感知蛋白- Prestin 10 1.4.1 Prestin 10 1.4.2 超音波刺激工程化聽覺感知蛋白 12 1.5 腦部基因轉殖 12 1.5.1 傳統基因遞送方式 12 1.5.2 超音波與基因負載微氣泡遞送基因 13 1.6 研究目的與內容 14 2 材料與方法 16 2.1 緒論 16 2.2 P-MBs 之製備 16 2.2.1 基因之製備 16 2.2.2 正電微氣泡之製備 16 2.2.3 負載聲敏感基因之微氣泡製備 18 2.2.4 光學定性分析 18 2.2.5 濃度、粒徑與電位量測分析 19 2.2.6 負載聲敏感基因之微氣泡的定量與負載效率 19 2.2.7 負載聲敏感基因之微氣泡之聲學穩定性 20 2.2.8 負載聲敏感基因之微氣泡之聲學擊破閾值 21 2.3 細胞實驗 23 2.3.1 人類神經母細胞瘤細胞培養與繼代 23 2.3.2 細胞膜通透性 23 2.3.3 細胞轉染實驗 25 2.4 動物實驗 25 2.4.1 動物實驗架構 26 2.4.2 超音波治療參數開啟血腦屏障之評估 27 2.4.3 血腦屏障開啟程度與傷害評估 28 2.4.4 超音波與負載聲敏感基因之微氣泡轉殖基因成效 29 2.4.5 腺病毒基因轉殖 29 2.5 類帕金森氏症動物模型治療 30 2.5.1 超音波治療流程 31 2.5.2 動物行為測試-beam walking test 31 2.5.3 動物行為測試-open field test 32 2.5.4 免疫染色分析 33 2.5.5 西方墨點法分析 35 2.6 統計分析 36 3 結果與討論 37 3.1 負載聲敏感基因之微氣泡之物理特性分析 37 3.1.1 電性轉變及光學分析 37 3.1.2 粒徑分析 38 3.1.3 基因負載效率 39 3.1.4 P-MBs之聲學穩定性實驗 40 3.1.5 負載聲敏感基因之微氣泡之聲學特性 41 3.2 細胞實驗 42 3.2.1 細胞膜通透性上升 42 3.2.2 細胞轉殖聲敏感基因之超音波最佳化參數 43 3.3 動物實驗 46 3.3.1 開啟血腦屏障之參數評估 47 3.3.2 超音波活體基因轉殖 48 3.3.3 腺病毒基因載體之表現量分析 51 3.3.4 Prestin與超音波刺激多巴胺細胞發生鈣離子流動 52 3.3.5 Beam walking test 之分析 55 3.3.6 Open field test 之分析結果 56 3.3.7 多巴胺神經修復成效 59 3.3.8 西方墨點分析法 60 4 結論與未來工作 63 4.1 結論 63 4.2 未來應用與發展 63 參考文獻 65

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