在目前的癌症治療中，由於長期接受藥物治療下所產生的抗藥性問題為癌症治療過程中最主要的阻礙之一，其主要機制可由多種模式控制，而其中最常見的方式為透過細胞膜表面過度表現的 p-glycoprotein 將進入細胞的藥物排出細胞外，該過程為耗能之主動運輸。有鑑於此，實驗中期望能夠利用外表修飾磁性奈米鐵之腺相關病毒 Ironized-AAV2，在外部磁場輔助下來達到改善抗藥性的問題，且具有控制病毒移動的功能，並同時提升病毒進入細胞的能力。實驗中將以綠色螢光蛋白 GFP 基因進行佐證及最佳化，來驗證此基因輸送系統確實得以改善抗藥性問題；進而作為攜帶基因 KillerRed，經轉錄轉譯後所表現之蛋白可作為光動力治療中所需之光敏劑。證明當腺相關病毒攜帶具有功能基因序列時，可以應用於不同治療中，本研究之實驗結果說明了腺相關病毒表面在修飾磁性奈米鐵後，可以透過外部磁場輔助下來控制病毒移動，亦可改善抗藥性問題並且同時應用於光動力治療中。此載體同時具備改善抗藥性問題、靶向性且可提升腺相關病毒所攜帶物質進入細胞之能力，由於可應用於光動力治療中，為一安全且具有高度發展潛力之輸送系統，並期許未來可應用於疾病及癌症治療中。

Recently, multidrug-resistant (MDR) is one of the major challenges in cancer therapy when the patients have been treated with the chemotherapy. Among potential factors associated with MDR, overexpression of P-glycoprotein (P-gp) has been commonly implicated in resistance to chemotherapeutic drugs. However, the pump drugs out of cancer cells of major mechanism triggered by P-gp overexpression has enhanced the survival rate of MDR cancer cells. In this study, we utilizes the adeno-associated virus serotype 2 (AAV2) chemically modified with iron oxide nanoparticles (~ 5 nm). ”Ironized-AAV2” displays a controllable viral distribution through a magnetic field (2000 – 4000 Gauss) and overcomes the pump drugs out of MDR human breast cancer MCF-7 cells with P-gp overexpression. Consequently, Ironized-AAV2 improved the cell uptake of viral particles compared with virus alone. Furthermore, a gene for production of the photosensitive protein KillerRed was introduced into the AAV2 genome to endow photo-dynamic therapy (PDT); or light-triggered oncolytic virotherapy (OV). In vitro experiments revealed that magnetic guidance of “ironized” AAV2-KillerRed in conjunction with PDT significantly decreases the cell viability of cancer cell via apoptosis. This proof-of-concept could be a treatment of MDR cancer by guided and highly localized micro-scale, light-triggered OV.

審定書…………………………………………………………………………. I
授權書…………………………………………………………………………. ii
致謝……………………………………………………………………………. iii
中文摘要………………………………………………………………………. iv
英文摘要………………………………………………………………………. v
目錄……………………………………………………………………………. vi
圖次……………………………………………………………………………. ix
表次……………………………………………………………………………. x

第 一 章 前言…...………………………………………………………….. 1
1-1 癌症治療...………………………………………………………….... 1
1-1-1概況...…………………………………………...………………. 1
1-1-2外科手術治療...……………………………………...…………. 1
1-1-3放射治療...…………………………………...…………………. 2
1-1-4化學藥物治療...………………………………...………………. 2
1-2多重抗藥性與常見之癌症……………………………………………. 4
1-2-1多重抗藥性…..…………………………………………………. 4
1-2-2乳癌……………………………………………………………... 6
1-3基因及藥物輸送系統…………………………………………………. 7
1-3-1腺相關病毒……………………………………………………... 9
1-3-2磁性奈米鐵粒子………………………………………………... 10
1-4光動力治療……………………………………………………………. 10
1-5研究動機與目的………………………………………………………. 12

第 二 章 材料及實驗方法…………………………………………………. 13
2-1 實驗材料……………………………………………………………... 13
2-2 實驗方法……………………………………………………………... 14
2-2-1細胞培養………………………………………………………... 14
2-2-2腺相關病毒AAV-2備製……………………………………….. 14
2-2-2-1高效能轉化大腸桿菌D5α之轉型作用…………………. 14
2-2-2-2質體 DNA之純化……………………………………….. 15
2-2-2-3腺相關病毒AAV2備製前期-共轉染……………………. 15
2-2-2-4腺相關病毒AAV2備製後期-病毒純化…………………. 16
2-2-2-5不同濃度 DOX 於普通及抗藥性細胞株之細胞活性及抗藥性檢測………………………………………………... 18
2-2-3材料合成………………………………………………………... 19
2-2-3-1磁性奈米鐵粒子修飾之腺相關病毒 Ironized-AAV2….. 19
2-2-3-2材料物性測試…………………………………………….. 19
2-2-3-3材料毒性測試…………………………………………….. 20
2-2-3-4備製比例最佳化I…………………………………………20
2-2-3-5備製比例最佳化II及局部磁吸控制病毒移動之檢測….. 21
2-2-3-6不同磁吸方式對於基因表現之影響及比較…………….. 21
2-2-4磁性奈米鐵粒子修飾之腺相關病毒 Ironized-AAV2 於光動治療之應用……………………………………………………. 22
2-2-4-1細胞之光照時間耐受度及最佳化……………………….. 22
2-2-4-2 KillerRed 紅色螢光蛋白表現與光源照射後之紅色螢
光蛋白之定性觀察及細胞活性之檢測之測試…………... 22
2-2-4-3 KillerRed 蛋白表現並比較光照前後活細胞與死細胞之檢測…………………………………………………... 23
2-2-4-4 KillerRed 蛋白表現並觀察光照後活性氧化物質 (ROS) 之產生情形…………………………………….. 23
2-2-4-5 KillerRed 蛋白表現並比較光照前後細胞凋亡情形…... 24
2-2-4-6 Ironized-AAV2(KR) 之移動並觀察光照後細胞凋亡及活細胞與死細胞之檢測情形…………………………... 25

第 三 章 結果與討論………………………………………………………. 27
3-1腺相關病毒AAV-2備製及抗藥性細胞株之抗藥性檢測……………27
3-1-1高效能轉化大腸桿菌 D5α之轉型作用………………………. 27
3-1-2質體 DNA 純化後之濃度及功能檢測………………………... 27
3-1-3腺相關病毒AAV2備製…………………………………………30
3-1-4不同濃度 DOX 於普通細胞株及抗藥性細胞株之細胞活性比較……………………………………………………………. 31
3-2磁性奈米鐵與腺相關病毒之合成……………………………………. 33
3-2-1磁性奈米鐵粒子修飾之腺相關病毒之物性檢測……….. 33
3-2-2材料毒性測試…………………………………………………... 34
3-2-3磁性奈米鐵粒子：EDC之合成比例最佳化……………………35
3-2-4不同磁吸方式對於基因表現之影響及比較…………………... 38
3-3磁性奈米鐵粒子修飾之腺相關病毒 Ironized-AAV2 於光動力治療之應用…………………………………………………………... 40
3-3-1細胞之光照時間耐受度及最佳化……………………………... 40
3-3-2交替磁場輔助下 KillerRed 紅色螢光蛋白表現之測試……... 41
3-3-3外部磁場輔助下 KillerRed 蛋白光源照射後之紅色螢光蛋白之定性觀察及細胞活性之檢測……………………………. 42
3-3-4外部磁場輔助下提升 KillerRed 蛋白表現並比較光照前後活細胞與死細胞之檢測………………………………………. 44
3-3-5外部磁場輔助下提升 KillerRed 蛋白表現並觀察光照後活性氧化物質 (ROS) 之產生情形…………………………….. 47
3-3-6外部磁場輔助下提升 KillerRed 蛋白表現並比較光照前後細胞凋亡情形…………………………………………………. 48
3-3-7外部磁場輔助下控制 Ironized-AAV2(KR) 之移動並觀察光照後細胞凋亡及活細胞與死細胞之檢測情形………………. 51

第 四 章 結論……………………………………………... 53
參考文獻………………………………………………………………... 54

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