基因轉染技術可透過調控細胞內基因表現而改變細胞生長、遷移或凋亡以應用於疾病治療。過去十年來，人類間質幹細胞 (hMSCs)在腫瘤治療與再生醫學領域的應用極受矚目，結合基因轉染技術將可進一步地調控幹細胞基因表達而獲取更佳的醫學應用效果。其中開發可針對hMSCs進行安全且高效率的基因轉染工具是一項重要課題。
本研究開發高分子-氧化鐵奈米粒子之複合材料作(PNT)作為hMSCs之基因磁轉染系統，此材料組成包括γ-polyglutamic acid (γ-PGA)修飾之氧化鐵奈米粒子(γ-PGA-SPIONs)、poly (β-amino esters) (PAE)以及質體DNA (pDNA)。SPIONs透過熱裂解法合成，而γPGA-SPIONs則以配基交換方法製備。以SQUID分析其飽和磁化率可達39.8 emμ/g，且於MRI T2影像中具備良好之對比效果(r2 = 334.7 mM-1s-1)。本研究中所合成之PAE於高分子/核酸重量比(polymer/pDNA weight ratio) 20以上時可有效攜載pDNA，透過靜電作用力與γPGA-SPIONs形成PNT複合基因載體可對hMSCs進行基因轉染。實驗中針對影響基因轉染效率的重要參數進行探討，包括：PAE/pDNA重量比、載體PNT稀釋倍數，以及 γPGA-SPIONs用量。於強力磁鐵吸引下，PNT載體可大幅增進hMSCs細胞的轉染效率。在無血清的環境下，PNT其磁轉染效率較PAE高分子基因載體高出三倍以上。經流式細胞儀偵測PNT於hMSCs之磁轉染效率可達70%以上，遠高於市售之轉染試劑，且於此條件下細胞存活率可維持90%以上。在含血清(10% FBS)的環境下，PNT其磁轉染效率相較於PAE或PNT無外加磁場吸引組別具有更佳的基因轉染效率。此外，hMSCs細胞經由PNT磁轉染後不影響其硬骨分化表潛力或腫瘤趨向遷移能力。
本研究針對所開發的PNT磁轉染系統，探討兩種重要的生醫應用，包括：1. 腫瘤治療與2. 幹細胞定向分化誘導。

1. 腫瘤治療應用：利用所開發之PNT磁轉染技術促使hMSCs細胞大量表達腫瘤壞死相關之誘導凋亡因子(tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)。本研究使用ELISA assay分析TRAIL protein的表現，PNT磁轉染相較於市售Lipofectamine 2000可提高hMSCs的TRAIL蛋白質表約五倍 (18.0 ng/mg → 86.3 ng/mL)。細胞共培養實驗結果指出表達TRAIL的hMSCs可有效誘導HeLa癌細胞凋亡。
2. 誘導幹細胞定向分化：將可促進hMSCs進行軟骨分化之TGF-β或Sox9基因以PNT磁轉染系統傳輸至hMSCs。相較於使用Lipofectamine 2k進行基因轉染，PNT磁轉染可提高TGF-β與Sox9表達量相較分別約五倍與四倍。經TGF-β或Sox9之磁轉染四週後，hMSCs細胞團塊切片染色上可觀察到明顯Alcian blue呈色反應，其collagen II專一性免疫螢光染色也具顯著表達。生化定量結果顯示TGF-β或Sox9磁轉染可提高hMSCs的葡萄糖胺(Glycosaminoglycans, GAGs)表達量約兩倍以上；而Collagen的表現量也提高了約四倍。

綜合以上結果，本研究所開發之PNT複合式基因載體搭配磁轉染技術可成功應用於hMSCs細胞的基因傳輸應用。其具有低細胞毒性，高效率以及可大量生產等優點，我們預期以PNT改造之hMSCs未來在腫瘤治療與再生醫學上均具有高度應用潛力。

Gene transfection is the technique capable of directly manipulating cellular gene expression, which can be applied for disease treatment via regulating cell physiology. In the past decade, human mesenchymal stem cells (hMSCs) have received great attention for their enormous potential on cancer therapy and regenerative medicine. Applications of gene transfection techniques on hMSCs further advanced their medical applications. One of the key tasks for such integration is the development of safe and efficient gene delivery tools for hMSCs.
In this study, we proposed a polymer/superparamagnetic iron oxide nanoparticles (SPIONs) polyplex (PNT) system, comprising of γ-poly (glutamic acid) (γPGA)-modified SPIONs (γPGA-SPIONs), poly (β-amino esters) (PAE), and plasmid DNA (pDNA) for efficient magnetically-assisted gene delivery (magnetofection) to hMSCs. SPIONs were prepared using thermal decomposition method, and γPGA-SPIONs were synthesized using a ligand exchange process. The magnetization of γPGA-SPIONs was up to 39.8 emμ/g measured by SQUID, which showed significant contrast enhancement on MRI T2-weighed imaging (r2 value = 334.7 mM-1s-1) of hMSCs. To prepare PNT system, PAE was used to fully condensed pDNA at or above the weight ratio (PAE pDNA) of 20. Afterwards, the polyplexes were combined with γPGA-SPIONs via electrostatic interactions to form PNT. PNT-mediated magnetofection efficiency was optimized by studying several key transfection parameters, including: polymer/pDNA weight ratio, polymer dilution factor and amount of γPGA-SPIONs. The transfection efficiency of PNT was greatly enhanced by applying with an external magnetic attraction. Under optimized magnetofection conditions, comparing to PAE polyplexes, PNT increased cellular uptake of pDNA up to 3-fold under serum-free condition. Additionally, PNT showed low cytotoxicity (viability ~ 90%) and exhibited excellent magnetofection efficiency (> 70%) on hMSCs compared to other commercial transfection agents. Additionally, PNT-mediated magnetofection did not cause detrimental effects on the osteogenic differentiation and tumor tropism of hMSCs.
In this study, we investigated the application potential of PNT magnetofection system on two important bioengineering aspects: (1) Cancer therapy and (2) Directing stem cell differentiation.

1. Cancer therapy: hMSCs were transfected to express tumor necrosis factor-related apoptosis inducing ligand (TRAIL) by using PNT magnetofection technique. TRAIL protein was successfully detected from the transfected hMSCs by ELISA assay. 5-fold increased on TRAIL expression was attained by PNT magnetofection compared to Lipofectamine 2000 (18.0 ng/mg → 86.3 ng/mL). The therapeutic potential of TRAIL-expressing hMSCs (TRAILhMSCs) for cancer therapy was explored on HeLa cells using an in vitro co-culture model. The results demonstrated that TRAILhMSCs could induce significant apoptosis on HeLa cells.

2. Directing stem cell chondrogenic differentiation: The genes known to promote stem cell chondrogensis, such as TGF-β and Sox9, were separately delivered using the PNT technique. Expression of TGF-β and Sox9 by PNT magnetofection was 5- and 4-fold higher than gene delivery by Lipofectamine 2000 respectively. After magnetofection for 4 weeks, the enhanced expression of chondrocyte-specific biomacromolecules, such as glycosaminoglycans (GAGs) and collagen II was observed from TGF-β- or Sox9-transfected hMSCs using Alcian blue staining and immunofluorescence staining. Finally, the expression amount of GAGs and collagen were quantitated by biochemical assay, showing around 2-fold increase of GAGs and 4-fold increase of collagen expression from TGF-β- or Sox9-transfected hMSCs compared to the control groups.

Taken together, we’ve successfully created a novel PNT Magnetofection system with excellent gene delivery efficiency and negligible cytotoxicity on hMSCs. Our current results suggest that the PNT-mediated genetically-engineered hMSCs possesses great potential on both cancer therapy and tissue regeneration.

摘要 2
Abstract 4
目錄 6
圖目錄 12
表目錄 17
第一章、緒論 18
1.1前言 18
1.2研究動機與目標 18
第二章、文獻回顧 21
2.1基因載體 21
2.2.1基因傳遞與治療系統 21
2.2.2病毒載體 23
2.2.3非病毒載體 26
2.2超順磁氧化鐵奈米粒子 29
2.2.1奈米粒子之特性與應用 29
2.2.2氧化鐵奈米粒子於基因傳遞之應用 30
2.3人類間質幹細胞 33
2.4 Tumor Necrosis Factor-related Apoptosis-inducing Ligand 35
2.4.1細胞凋亡途徑(Cell apoptosis pathway) 35
2.4.2 TRAIL protein與其訊號傳遞途徑 37
2.4.3腫瘤治療之應用與限制 39
2.4.4延長半衰期與增進活體治療效率之策略 39
2.5軟骨分化於組織修復應用 40
2.5.1全球人口老化 40
2.5.2骨關節炎與其治療 42
2.5.3間質幹細胞於軟骨分化策略 43
2.6基因傳遞系統於間質幹細胞概況 47
第三章、實驗材料與方法 49
3.1實驗材料(I) 49
3.1實驗材料(II) 50
3.2載體製備與分析方法 51
3.2.1 SPIONs合成與純化 51
3.2.2 γPGA-SPIONs製備與純化 51
3.2.3氧化鐵奈米粒子之基本特性分析 51
3.2.4 PAE與PNT polyplexes載體製備 52
3.2.5膠體電泳分析 52
3.2.6穿透式電子顯微鏡觀察 52
3.2.7載體之粒徑大小與穩定性測試 52
3.3 PNT polyplexes於hMSCs之磁轉染與分析 53
3.3.1細胞培養與繼代 53
3.3.2基因傳遞與轉染效率分析 53
3.3.3磁轉染對細胞毒性之測試 53
3.3.4普魯士藍染色鑑定 54
3.3.5質體DNA攝入含量分析 54
3.3.6核磁共振造影 54
3.3.7細胞骨架與細胞核染色 54
3.3.8硬骨分化測試 54
3.3.8.1茜素紅S染色 55
3.3.8.2 Osteonectin 基因表現分析 55
3.4 TRAIL protein分析與癌細胞毒殺實驗 56
3.4.1酵素連結免疫吸附法分析 56
3.4.2 GFPHeLa共培養 57
3.4.3 LucHeLa共培養 57
3.4.4細胞凋亡caspase 3 分析 57
3.4.5腫瘤趨向性探討 58
3.6軟骨分化 58
3.6.1 TGF-β與Sox9基因傳遞與表現 58
3.6.2 3D pellet culture促進軟骨分化 58
3.6.3樣品包埋、切片與染色 59
3.6.3.1 Alcian blue染色 60
3.6.3.2 Collagen type II免疫螢光染色 60
3.6.4 Papain digestion與特徵蛋白質分析 60
3.6.4.1 DNA assay測試 61
3.6.4.2 GAG assay測試 61
3.6.4.3 Collagen assay測試 62
3.7數據分析 63
第四章、實驗結果與討論 64
第一部分、磁轉染於hMSCs細胞之探討 64
4.1氧化鐵奈米粒子與基因載體之特性分析 64
4.1.1 SPIONs與γPGA-SPIONs基本鑑定 64
4.1.2膠體電泳分析 66
4.1.3 PNT載體之粒徑大小分析 66
4.1.5載體之穩定性觀測 68
4.2 PNT磁轉染對轉染效率與細胞毒性之探討 69
4.2.1載體稀釋倍數之影響 69
4.2.2 hMSCs細胞代數 73
4.2.3載體與pDNA之重量比 75
4.2.4 γPGA-SPIONs含量 77
4.2.5 γ-glutamyl transpeptidase抑制測試 80
4.2.6 PNT polyplexes於血清環境之磁轉染 82
4.2.6最佳化之轉染效率與毒性分析 82
4.3染色與影像鑑定 84
4.3.1 細胞骨架與細胞核染色 84
4.3.2普魯士藍染色鑑定 85
4.3.3 hMSCs細胞於MRI T2之影像 86
4.3.4 TOTO-3螢光分析 87
4.4磁轉染對硬骨分化之探討 88
4.4.1茜素紅S染色 88
4.4.2硬骨之特徵基因表現 88
第二部分、磁轉染於癌症治療之應用 90
4.5 TRAIL protein表現與毒殺癌細胞 90
4.5.1 TRAIL protein定量分析 90
4.5.2 GFPHeLa cells共培養之定性觀察 91
4.5.3 LucHeLa cells共培養之定量分析 92
4.5.4凋亡特性Caspase 3 之偵測 93
4.5.5腫瘤趨向性之探討 93
第三部分、磁轉染於軟骨分化之應用 95
4.7軟骨分化之探討 95
4.7.1 TGF-β與Sox9磁轉染之毒性分析 95
4.7.2轉染效率與細胞毒性於6-well系統之測試 95
4.7.3 TGF-β與Sox9基因表現分析 98
4.7.4 Alcian blue染色鑑定 98
4.7.5 Collagen II之免疫螢光染色 99
4.7.6 DNA assay 100
4.7.7 GAG assay 101
4.7.8 Collagen assay 102
第五章、結論 104
第六章、未來展望 105
第七章、附錄 106
開發複合式高分子/氧化鐵奈米載體作為具氧化還原敏感之基因傳遞平台 106
7.1摘要 106
7.2材料與方法 108
7.2.1 SPIONs合成與citric acid修飾 108
7.2.2 CA-SPIONs基本特性分析 108
7.2.3 PDS合成 108
7.2.4 PEI-SH合成 108
7.2.5 SPION-PDS合成 109
7.2.6 NSP載體合成 109
7.2.7膠體電泳分析 109
7.2.8 GSH還原與pDNA釋放之測試 109
7.2.9細胞轉染測試 109
7.3結果與討論 110
7.3.1 CA-SPIONs基本特性分析 110
3.7.3 NSP載體鑑定 112
3.7.4 GSH敏感測試 114
7.3.5 NSP於HEK293T之細胞轉染 115
7.4結論與未來展望 116
參考文獻 117

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