惡性腫瘤常居國人十大死因之首，其中又以腦癌最為惡劣，五年存活率不及一成。而血腦屏障與細胞抗藥性使得化療與基因治療等療法效果不彰。本研究將針對腦癌細胞之抗藥性評估而設計一快速檢測系統，利用Qβ 類病毒粒子 - 金奈米簇生物礦化之複合材料實現之。且搭配智慧型手機結合雲端運算評估癌細胞抗藥性，將有助於選擇後續治療手段。在治療方面，本研究研發三項主要解決方案。若針對無明顯抗藥性腫瘤，採用對流輸送增強法 (convection-enhanced delivery, CED) 直接將裝載化療藥物Epirubicin (EPI) 之 Qβ 類病毒粒子送入腫瘤進行原位治療。若針對抗藥性較強之腫瘤，本研究設計了兩套 RNAi 輸送系統以降低其抗藥性，目前也已經成功利用基因工程技術一步合成自動封裝第 1 代雙功能 RNAi 骨架於內部的 Qβ 類病毒載體，並可於載體表面修飾增強細胞吞噬多肽 (cell penetration peptide, CPP) 以及協助穿越血腦屏障的 Apolipoprotein E (ApoE) 多肽。類病毒載體不具細胞毒性，可利用 E. coli. 快速、方便、大量生產，大幅降低基因藥物製造成本與提升安全性。針對造成 Temozolomide (TMZ) 抗藥性的 c-MET 進行 RNA 干擾治療，提升 TMZ 治療效果。此外，本研究進一步設計出第二代，包含兩種 RNAi 功能與螢光追蹤功能且能自動封裝的多功能的 RNAi 骨架，突破目前無法及時定量追蹤 RNAi 的困境。此骨架結合三向結構域 (three way junction domain)，可實現多重 RNAi 輸送，並同時追蹤 RNA 之分解現象，此多重 RNAi 輸送也提供多重基因治療的可能。最後，本研究將以實際動物實驗進行最佳化劑量、治療效能及存活率評估。結合上述研究，此整合式自組裝多功能類病毒系統可望減低嘗試藥物治療之時間與資源、減少抗藥性及提升輸送效率，期望能提升腦癌之治療效率與提升病患之存活。

Malignant tumors often rank the top ten causes of death in Taiwan. Brain tumor is known as the most lethal cancer which the five-year survival rate is less than 10%. The drug resistance and blood-brain barrier (BBB) makes chemotherapy and gene therapy less effective. This study will design a rapid detection system for drug resistance evaluation of brain cancer cells, comprising Qβ-like viral gold nano-cluster composite materials, smart phones and cloud computing to help make a strategy of follow-up treatment. In terms of treatment, this study developed three main solutions. For the low drug-resistant tumors, we developed Epirubicin-loaded virus-like particles (VLPs) which can be delivered to brain tumor by convection-enhanced delivery (CED) for in situ treatment. The results clearly indicated that a combination of Epirubicin-loaded VLPs and CED can serve as a flexible and powerful synergistic treatment in brain tumors without evidence of systemic toxicity.
For drug-resistant tumors, we have successfully developed the first-generation bifunctional RNAi backbone (i.e., c-MET RNAi), which can be automatically encapsulated in VLPs to downregulate c-MET expression and inhibit the DNA repair mechanism (i.e., demethylation) for promoting the curative efficacy of oral Temozolomide (TMZ). Furthermore, the surface of VLPs is further modified with enhance cell phagocytic peptides (CPP) and Apolipoprotein E (ApoE) peptides to enhance the efficacy of cell uptake and BBB penetration. The results of this study could be critical for the design of RNAi-based genetic therapeutics for promoting chemotherapy against brain tumors.
Furthermore, we developed second-generation multi-functional RNAi scaffold based on three-way junction (3WJ) domain structure with two RNAi functions and real-time fluorescence tracking function to overcome the current dilemma that RNAi cannot be tracked quantitatively in time in cells. This study can achieve multiple RNAi delivery and simultaneously track the decomposition of RNAi in cells to provide the possibility of multiple gene therapy. The results demonstrated that more than 95% of the tumors were suppressed after the combined treatment (gene therapy + radiotherapy), and 100% of the animals survived, compared with the current clinical radiotherapy using carboplatin as radio-sensitizer (only 80% of the final animal survived). Summarizing the above research, this self-assemble multi-functional viral-based system is expected to reduce the drug resistance and enhance drug delivery efficiency that may improve the treatment efficiency and the survival of patients with brain tumor.

目錄
論文審定書 i
誌謝 ii
中文摘要 iv
Abstract v
目錄 vii
圖次 xiii
表次 xvi
第1章、 緒論 1
1.1. 研究背景與動機 1
1.2. 腦癌現行治療困境與多功能平台需求 3
1.2.1. 腦癌之流行病學與疾病概述 3
1.2.2. 腦癌之治療手段比較與困境 4
1.2.3. 腦癌治療之多功能平台需求 6
1.3. 類病毒奈米粒子 (virus-like particles, VLPs) 8
1.3.1. VLPs 簡介 8
1.3.2. VLPs 現今應用範圍 10
(1) 作為疫苗或疫苗載體 10
(2) 作為藥物輸送載體 11
(3) 作為基因輸送載體 13
(4) 結合金屬或其他成分的整合應用 13
1.3.3. VLPs 應用之瓶頸與作為平台之可能性 14
1.4. 腦癌細胞之抗藥性評估 15
1.4.1. 癌細胞抗藥性的產生與細胞內氧化壓力 15
1.4.2. 現行檢測 GSH 方法之優缺點比較 16
1.4.3. 紙基材的生物感測器優勢 18
1.4.4. 蛋白質-奈米簇用於檢測 19
1.5. 血腦屏障與對流增強輸送法 22
1.5.1. CED 簡介與血腦屏障 22
1.5.2. 泛艾黴素之化學治療與輸送 23
1.5.3. CED 搭配 VLP 作為藥物輸送追蹤平台之可行性 24
1.6. 基因治療 26
1.6.1. RNA干擾之原理簡介 26
1.6.2. RNAi 相關輸送方式與面臨困境 28
1.6.3. RNA骨架之摺疊模式與 RNAi 之影響 30
1.6.4. 表皮生長因子與 let-7g miRNA 作為基因治療標的 32
1.6.5. RNA 螢光適體與即時RNA追蹤 34
1.6.6. 多功能 RNA 在 VLPs 中自組裝的可行性分析 36
1.7. 研究目的 37
第2章、 研究設計 40
2.1. 研究架構設計 40
2.2. 實驗材料 43
(1) 質體、核酸與酶 43
(2) 化學藥品與實驗套裝 44
(3) 耗材用品 48
2.3. 實驗儀器 49
第3章、 利用金奈米簇生物礦化 VLPs 作為 GSH 生物感測器 (應用一) 51
3.1. 研究簡介 51
3.2. 研究方法 52
3.2.1. VLPs 金奈米簇製作 52
3.2.2. 穿透式電子顯微鏡 52
3.2.3. 蔗糖濃度梯度超高速離心影像 52
3.2.4. AuVCs螢光影像 52
3.2.5. AuVCs的螢光光譜測量 53
3.2.6. AuVCs 對 H2O2/TMB 的動力學參數 53
3.2.7. 側向流道試紙的製備 53
3.2.8. 使用 LFPB 進行 GSH 檢測 53
3.2.9. LFPB 的穩定性研究 54
3.2.10. 抗干擾與回收率測試 54
3.2.11. 細胞實驗 55
3.2.12. 統計分析 55
3.3. 研究結果與討論 55
3.3.1. VLPs 和 AuVCs 的製造與鑑定 57
3.3.2. AuVCs 的動力學和穩定性研究 59
3.3.3. 使用 AuVCs 以 H2O2/TMB 進行 GSH 比色檢測 62
3.3.4. AuVCs 催化金粒子生成之訊號產生機制 66
3.3.5. 使用側流式等離子生物感測器 (lateral flow plasmonic biosensor，LFPB) 測定 GSH 68
3.3.6. 使用 LFPB 測定細胞內和組織之 GSH 含量並預測抗藥性 75
3.4. 總結 78
第4章、 VLPs 包覆 EPI 並以 CED 輸送進行腦癌治療 (應用二) 79
4.1. 研究簡介 79
4.2. 研究方法 80
4.2.1. Qβ衣殼蛋白 (QβCP) 和綠色螢光蛋白共表達質體 80
4.2.2. CPP修飾 VLPs gVLPs 81
4.2.3. 將 EPI 包裝於 CPP-gVLPs 中之包覆率測試 82
4.2.4. 穿透式電子顯微鏡 82
4.2.5. EPI釋放曲線測定 83
4.2.6. 細胞實驗 83
4.2.7. CPP-gVLPs通過尾靜脈注射和 CED 注射的生物分佈 84
4.2.8. 動物實驗：腦部腫瘤植入 85
4.2.9. CED 注射實驗 86
4.2.10. 組織病理學研究 86
4.2.11. CED 注入的EPI @ CPP-gVLPs的抗腫瘤效率 86
4.2.12. 血液生化分析和免疫測定 87
4.3. 研究結果與討論 87
4.3.1. EPI@CPP-gVLPs 的生產和鑑定 87
4.3.2. 體外細胞攝取和細胞毒性研究 94
4.3.3. CPP-gVLPs的體內生物分佈分析 96
4.3.4. CED 的組織毒性分析和腦內藥物分佈 99
4.3.5. 以 CED 注射 EPI@CPP-gVLPs 的動物體內抗腫瘤效率 103
4.3.6. 血液生化分析 106
4.4. 總結 107
第5章、 包裝多功能 RNAi 骨架之 VLPs 偕同 CED 進行腦癌治療 (應用三) 109
5.1. 研究簡介 109
5.2. 研究方法 115
5.2.1. 質體建構 115
5.2.2. VLPs菌內表現與純化 115
5.2.3. 菌體全RNA與VLPs內部RNA之純化 115
5.2.4. VLPs性質鑑定 116
5.2.5. VLPs 穩定性實驗 117
5.2.6. 細胞攝取實驗 117
5.2.7. 北方墨點法 (Northern blotting) 117
5.2.8. 西方墨點法 118
5.2.9. 螢火蟲螢光素酶表現檢測 119
5.2.10. 免疫螢光測定 119
5.2.11. 細胞生長測定 119
5.2.12. 細胞侵襲與遷徙能力測定 120
5.2.13. VLPs細胞毒性測試 120
5.2.14. 動物實驗 121
(1) 動物實驗程序 121
(2) 體內腫瘤抑制研究 121
(3) 組織病理學研究 121
5.3. 研究結果與討論 122
5.3.1. 多功能 RNAi 骨架的結構設計與表達系統構建 122
5.3.2. 多功能 RNAi 骨架受到 Qβ 衣殼保護提升穩定性 132
5.3.3. rQβ@b-3WJsiEGFR siLUC 可被細胞吞食並進行 RNAi 追蹤 135
5.3.4. RNAi 骨架包裝於 VLPs 可在 GBM 細胞進行多目標沉默 141
5.3.5. CED 注射體內腫瘤抑制 149
5.4. 總結 154
第6章、 綜合討論 156
6.1. 科學研究應用討論 157
6.2. 臨床應用討論 160
6.3. 延伸研究與未來發展方向 163
第7章、 結論 167
參考文獻 171
附錄 189
附錄一、歷年發表期刊論文 189
附錄二、歷年參與研討會發表 192
附錄三、歷年獲獎 193

 
圖次
圖 1 1、腦癌分類相對應之發展歷程圖與相關的基因異常。 4
圖 1 2、應用不同種類之病毒製作之 VLPs 衣殼模式3D模擬圖13。 9
圖 1 3、Qβ VLPs 之形成與結構特性。 10
圖 1 4、蛋白質輸送 VLPs 的生成和作用方式。 12
圖1 5、Qβ VLPs 之基本性質、常見應用與其優勢示意圖。 15
圖 1 6、金奈米簇結合的HEV奈米衣殼的3D建模。 20
圖 1 7、Qβ 衣殼蛋白之結合局部結構。 21
圖 1 8、CED的配置示意圖。 23
圖 1 9、RNAi 作用機制圖。 27
圖 1 10、Phi-29 噬菌體的 DNA 包裝馬達 3D 模擬圖。 31
圖 1 11、Broccoli 螢光 RNA 適體之篩選與結構應用示意圖。 35
圖 2 1、研究架構示意圖。 41
圖 3 1、GSH 生物感測器系統運作之概念圖。 56
圖 3 2、AuVCs 的特性鑑定。 58
圖 3 3、在相同參數下合成的AuVC和BSA-AuNC的比較。 59
圖 3 4、AuVCs 催化特性與酵素動力學鑑定。 61
圖 3 5、以DLS測量用 5 mM GSH 處理前後的 VLPs 之粒徑分佈。 63
圖 3 6、VLPs 之分解現象分析。 64
圖 3 7、利用 AuVCs 作為 GSH 檢測之探針與校正曲線之測定。 65
圖 3 8、HAuCl4 與不同基團反應的 UV-Vis 吸收光譜和相應的照片。 67
圖 3 9、AuVCs 作為金奈米粒子之生長模板之可行性分析。 68
圖 3 10、LFPB GSH 自動分析的圖像處理流程。 69
圖 3 11、LFPB 檢測區 (Y 區) 之局部SEM影像以及EDS元素分析。 71
圖 3 12、LFPB 之運作與判讀。 72
圖 3 13、AuVCs 與LFBP系統的穩定性測試。 74
圖 3 14、LFPB 使用 TMB 作為訊號源測試。 75
圖 3 15、以 LFPB 系統結合自動化判讀軟體進行細胞與真實組織檢體的 GSH 濃度測定，以及抗藥性評估。 77
圖 4 1、研究設計與研究流程示意圖。 80
圖 4 2、CPP 修飾 VLPs 之步驟簡圖。 82
圖 4 3、EPI 透過核酸親和力吸附的方式進入 VLPs 之模擬影像。 88
圖 4 4、EPI 之包覆途徑確認實驗。 90
圖 4 5、VLPs之包裝特性。 92
圖 4 6、CPP修飾與穩定性分析。 93
圖 4 7、CPP-VLPs 的細胞攝取效率分析。 95
圖 4 8、VLPs為載體之EPI毒性測試。 96
圖 4 9、注射 68Ga-DOTA 標記的 CPP-gVLPs 後原位腦腫瘤模型的體內 PET/CT 成像。 98
圖 4 10、CED 注射後的組織病理學分析。 101
圖 4 11、使用螢光顯微鏡觀察 CED 注射游離態 EPI 或 EPI@CPP-gVLPs 3小時之後在大腦中的分佈和腫瘤攝取情形。 102
圖 4 12、以 CED 注射方式評估 EPI@CPP-gVLPs的治療方案。 104
圖 4 13、利用 CED 輸送 EPI@CPP-gVLPs 之腫瘤抑制效率與存活率。 106
圖 4 14、CED 注射 CPP-gVLPs 7 天後動物的血液生化分析。 107
圖 5 1、單 RNAi 輸送前導研究之概念示意圖。 110
圖 5 2、多功能 VLPs 用於降低腦癌抗藥性之基因治療機制圖。 111
圖 5 3、前導研究重要數據。 112
圖 5 4、多功能 RNAi 骨架設計圖與運作概念。 114
圖 5 5、第 2.1 代 b-3WJMG si RNAi 骨架的表達系統設計與鑑定。 123
圖 5 6、包裝b-3WJMG si之VLPs螢光鑑定。 124
圖 5 7、包裝有b-3WJMG si骨架之VLPs之多肽修飾。 125
圖 5 8、第 2.2 代 b-3WJsiEGFR siLUC RNAi 骨架的設計與鑑定。 127
圖 5 9、包有b-3WJsiEGFR siLUC 之VLPs的螢光鑑定。 128
圖 5 10、利用單分子光漂白法檢測單VLP內部所含 b-3WJsiEGFR siLUC 數量。 131
圖 5 11、RNA 包裝於 VLPs 內部的穩定性分析。 134
圖 5 12、TAT 肽修飾與否的 rQβ@b-3WJsiEGFR siLUC 的三維 U87-MG 細胞腫瘤球攝取影像。 136
圖 5 13、RNA 骨架細胞內降解鑑定。 138
圖 5 14、DFHBI-1T 活化 rQβ@b-3WJsiEGFR siLUC 培養的細胞即時影像。 139
圖 5 15、挑選時間局部放大 DFHBI-1T 活化後 rQβ@b-3WJsiEGFR siLUC 處理的細胞初始 (0分鐘) 與40 分鐘的即時影像。 140
圖 5 16、rQβ@b-3WJsiEGFR siLUC 之 RNAi 功能驗證。 143
圖 5 17 、rQβ@b-3WJsiEGFR siLUC 之細胞生長、侵襲能力抑制驗證。 144
圖 5 18、Qβ@b-3WJsiEGFR let-7g的 RNAi 功能性試驗。 147
圖 5 19、EGFR 抑制和輸送 let-7g 導致 NFκB 抑制的可能機制。 148
圖 5 20、動物腦部以 CED 注射 Qβ@b-3WJsiEGFR let-7g 之螢光追蹤結果。 149
圖 5 21、注射 Qβ@b-3WJsiEGFR let-7g 治療後小鼠的腦 MRI 圖像。 151
圖 5 22、動物實驗隻小鼠腫瘤成長速率與動物存活率。 153
圖 6 1、利用 VLPs 作為多功能整合型治療應用平台的概念圖。 166
圖 7 1、本研究中自 Qβ 噬菌體 VLPs 衍生出的各種延伸應用之概念圖。 167
 
表次
表 1 1、GSH 之檢測方法及比較表。 17
表 2 1、本研究各應用主題與對應之 VLPs 品系對照表。 42
表 3 1、以DLS測量用 5mM GSH 處理前後的 VLPs之粒徑與PI值。 63
表 3 2、使用 LFPB 進行 GSH 標準添加的回收率測試。 73
表 4 1、本研究中所使用的引子序列與對應的酶切位點 (restriction sites)。 81
表 4 2、以DLS測量 gVLPs、EPI@gVLPs、與 EPI@CPP-gVLPs 之粒徑。 91

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