近年來、基因療法 ( Gene therapy ) 用於治療癌症，已成為一種具展望性的治療策略於對抗肝癌 ( HCC ; hepatocellular carcinoma )。然而治療性核酸質體( Plasmid DNA ) 能否成功運輸至細胞內並調控癌細胞表現，是眾多研究所面臨的一大挑戰。因此在本研究中，我們利用已知對 HCC 具有標靶作用的 SP94 胜肽修飾脂質磷酸鈣奈米載體 ( Liposome Calcium Phosphate Nanoparticle ，LCPP-NPs ) 標靶肝癌細胞。藉由具有核定位訊號 ( Nuclear Localization Signal ， NLS ) 魚精蛋白 ( Protamine ) 以提高轉染效果，讓癌細胞高度表達特定蛋白表現。更進一步，我們使用奈米粒子搭載具有誘導細胞凋亡的基因片段-腫瘤壞死因子相關凋亡誘導配體- TRAIL( Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand )，專一性的運輸至肝癌細胞中進行基因治療，希望能藉此讓肝癌細胞進行細胞凋亡，達到治療癌症效果。總結來說，這個具腫瘤標靶控細胞基因表現，達到顯著的基因治療目的。

Gene therapy for cancer treatment has become the very promising approach of hepatocellular carcinoma (HCC). However, the therapeutic plasmid DNA delivery into cancer cell and regulate the expression is a challenge of many studies. We utilize SP94 peptide as targeted ligand modifies liposome calcium phosphate nanoparticles (LCP-NPs) to specifically target HCC. We also use protamine to improve transfection efficiency which contains several nuclear localization signal (NLS). Moreover, we want to deliver an apoptosis inducer gene -TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) to induce tumor cell apoptosis. These results demonstrated that targeting LCP-NPs can specifically delivery therapeutic plasmid DNA into HCC to regulate cancer cell expression.

摘要 I
Abstract II
圖目錄 VI
縮寫目錄 VIII
第一章、緒論 1
一、 研究背景 1
1.1 肝癌介紹與形成原因 1
1.2 肝臟結構與肝癌內部訊號傳遞機制 2
1.3 肝癌的治療方法 6
1.4 基因治療(Gene therapy) 7
二、 研究目的 17
第二章、實驗材料與方法 18
2.1 所用材料 18
2.2 細胞培養 18
2.3 動物實驗 19
2.4 製備LCPP-NPs及SP94胜肽表面改質 19
2.5 LCPP-NPs定性分析 20
2.6 SP94-LCPP NPs細胞攝入與分析魚精蛋白運輸Plasmid至細胞核能力 20
2.7 基因轉染率分析 21
2.8 細胞存活率分析 21
2.9 調控基因表現研究 22
2.10 結合蕾莎瓦與SP94-LCPP NPs傳遞TRAIL質體DNA之複合療法分析細胞存活率及對腫瘤發展影響 23
2.11 蘇木素-伊紅(H&E; Hematoxylin and Eosin) 染色 23
2.12 免疫螢光染色 24
2.13 統計分析方式 24
第三章、實驗結果與討論 25
3.1 裝載Plasmid DNA之SP94 靶向磷酸鈣奈米載體的製備與定性分析 25
3.2 觀察搭載魚精蛋白的SP94-LCPP-NPs 運送Plasmid DNA至肝癌細胞的攝取表現 27
3.3 使用SP94-LCPP-NPs 運送Luciferase plasmid至肝癌細胞並轉染Luciferase表現 32
3.4 SP94-LCPP-NPs 運送TRAIL plasmid調控肝癌細胞表現TRAIL並利用提升鈣離子濃度使肝癌細胞表面死亡受體DR5表現量增加 34
3.5 藉由SP94-LCPP-NPs 運送TRAIL plasmid至肝癌細胞降低細胞活性並誘發細胞凋亡 36
3.6 結合肝癌臨床藥物蕾莎瓦與SP94-LCPP-TRAIL-NPs降低肝癌細胞抗藥性並提高藥物敏感性 38
3.7 結合SP94-LCPP-TRAIL-NPs與臨床藥物蕾莎瓦偕同作用有效抑制小鼠肝癌腫瘤生長 40
3.8 SP94-LCPP-TRAIL-NPs傳遞TRAIL plasmid進而抑制肝癌轉移 43
3.9 SP94-LCPP-TRAIL-NPs藉由傳遞TRAIL plasmid提升腫瘤微環境TRAIL蛋白表現並誘導細胞凋亡 45
第四章、結論 50
第五章、致謝 52
第七章、參考資料 53

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