口服非病毒載體基因傳輸，被視為最具吸引力但同時也是最艱難的投遞路徑。幾丁聚醣(chitosan, CS)為天然、高生物相容性之多聚醣高分子，可和負電DNA形成微粒載體幫助DNA進入細胞核。然而，轉染效率 (transfection efficiency)偏低及環境中性pH值的使用限制，為CS/DNA載體系統的主要缺點。為改善以上限制，本研究利用聚乙烯亞胺(polyethyleneimine, PEI)修飾CS。PEI為帶正電且具有高轉染效率之基因傳遞載體，然而其細胞毒性限制其在生醫領域上的應用。本研究將短鏈PEI (500-800Da) 接枝於CS上合成的CS-grafted-PEI(CS-g-PEI)，不僅能有效降低PEI的細胞毒性同時還保有其優異的基因轉染效率。實驗內容分為兩個部分。第一部分延續前期的研究成果，製造顆粒粒徑約100 nm，表面電位約+47 mv 之CS-g-PEI/ pEGFP-N2奈米微粒，研究其於細胞的胞吞(endocytosis)機制與傳輸路徑。並藉由小鼠動物實驗，分析奈米微粒在口服投遞後的活體內分布情形(biodistribution)，以及驗證其所攜帶的綠色螢光蛋白質體(EGFP-plasmid)確實可在動物體內表達。第二部分為human insulin plasmid奈米微粒。驗證其在酸鹼環境中可以維持穩定結構。利用人類結腸腺癌單層細胞(Caco2 monolayer)證實奈米微粒具有穿透能力與討論其傳輸途徑。在糖尿病小鼠模型中，使用本載體系統投遞human insulin plasmid後，進行人類胰島素的藥物效應動力學(pharmacodynamics, PD)研究；同時由18F-FDG/PET實驗，研究在口服該奈米微粒後對血糖的利用情況。結果顯示，本載體系統可由口服投遞路徑成功在糖尿病小鼠體內中轉譯出人類胰島素，並有效抑制血糖長達8天。

關鍵字：非病毒載體、基因傳輸、聚乙烯亞胺接枝幾丁聚糖、口服、糖尿病

Chitosan (CS), a natural-origin cationic polysaccharide, is biodegradable, non-toxic and soft-tissue compatible. It has the potential to condense anionic DNA into a compact structure through electrostatic interactions. This effectively protects DNA from nuclease degradation. However, low transfection efficiency and a limited working pH range (pH~6) of such binary CS/DNA complex are known as two major disadvantages when undertaking in vivo. Polyethyleneimine (PEI) is one of the most effective polymer-based gene carriers due to its high proton buffering capacity. Grafting low-molecular weight PEI onto CS was shown to induce lower cytotoxicity and was able to transfect a variety of cells in vitro comparably. We developed a system that has the best transfection efficiency with limited toxicity. The CS-g-PEI/pEGFP-N2 nanoparticle synthesized is about 100 nm in diameter and +47 mV in zeta potential. Endocytosis pathway and transport pathway are surveyed in this research. In vivo study showed the accumulating of NPs in liver of mice. Replace pEGFP-N2 with pCMV6-human insulin plasmid. NPs can stay integrity in alkaline solution and in the passing process of Caco2 monolayer. Diabetes mice model indicate CS-g-PEI/ pCMV6-human insulin NPs have an effect of reducing blood glucose and increasing the secreting of human insulin in liver, peyer’s patch, and kidney, which can keep the diabetes mice’s blood glucose stay in normal level up to 8 days.

Keywords: non-viral vector, gene transfection, CS-g-PEI, oral delivery, diabetes therapy

目錄 II
圖目錄 V
表目錄 VII
一、緒論 1
1.1 基因相關疾病 (Gene Related diseases) 1
1.2 基因治療 (gene therapy) 1
1.3 基因傳遞方式與系統 2
1.4 幾丁聚醣 (chitosan, CS) 4
1.5 利用幾丁聚醣及聚乙烯亞胺作為基因傳遞載體 4
1.6 研究目的與動機 6
二、材料與方法 7
2.1 材料 7
2.2 CS-g-PEI的合成、結構鑑定以及化學性質分析 7
2.2.1 CS-g-PEI的合成與結構鑑定 7
2.2.2 酸鹼滴定實驗 8
2.2.3 CS-g-PEI的降解測試 8
2.3 製備CS-g-PEI/pEGFP-N2奈米微粒 8
2.4 CS-g-PEI/pEGFP-N2奈米微粒的細胞內化(internalization)與胞吞路徑 9
2.4.1 製備螢光標定的pGL4.13 9
2.4.2 CS-g-PEI/pEGFP-N2奈米微粒的胞內化 9
2.4.3 CS-g-PEI/pEGFP-N2奈米微粒的胞吞路徑 9
2.5 CS-g-PEI/pEGFP-N2奈米微粒通過人類結腸腺癌單層細胞探討 10
2.5.1 製備螢光標定的CS-g-PEI 10
2.5.2 CS-g-PEI/pEGFP-N2奈米微粒通過Caco2單層細胞測定 10
2.6 CS-g-PEI/pEGFP-N2奈米微粒動物實驗 10
2.7 製備CS-g-PEI/pCMV6-human insulin奈米微粒 11
2.8 CS-g-PEI/pCMV6-human insulin奈米微粒透射電子顯微鏡(TEM)圖像 11
2.9 CS-g-PEI/pCMV6-human insulin奈米微粒pH穩定性測試 12
2.10 螢光能量共振轉移(fluorescence resonance energy transfer)測試 12
2.10.1 製備螢光標定的pGL4.13及螢光標定的CS-g-PEI 12
2.10.2 CS-g-PEI/pCMV6-human insulin奈米微粒通過Caco2單層細胞完整性 12
2.11 CS-g-PEI/pCMV6-human insulin奈米微粒的轉運途徑 13
2.12 CS-g-PEI/pCMV6-human insulin奈米微粒動物實驗 13
2.12.1 糖尿病鼠模型的建立 13
2.12.2 藥物效應動力學(pharmacodynamics, PD)測試 13
2.13 人類胰島素訊號在小鼠各個器官的分佈情況 14
2.14 正子造影技術分析血糖利用率 14
三、結果與討論 15
3.1 分析CS-g-PEI的合成、結構鑑定以及化學性質分析 15
3.1.1 1H NMR、FT-IR鑑定CS-g-PEI 15
3.1.2 酸鹼滴定 16
3.1.3 CS-g-PEI的降解測試 17
3.2 CS-g-PEI/pEGFP-N2奈米微粒的細胞內化與胞吞路徑 18
3.2.1 CS-g-PEI/pEGFP-N2奈米微粒的粒子大小及界面電位 18
3.2.2 CS-g-PEI/pEGFP-N2奈米微粒的胞內化 19
3.2.3 CS-g-PEI/pEGFP-N2奈米微粒的胞吞路徑 20
3.3 CS-g-PEI/pEGFP-N2奈米微粒穿透人類結腸腺癌單層細胞的探討 22
3.4 CS-g-PEI/pEGFP-N2奈米微粒動物實驗 23
3.4.1 奈米微粒於生物體內分布(biodistribution) 23
3.4.2 Cy5標定的CS-g-PEI/pEGFP-N2奈米微粒生物體器官冷凍切片 24
3.4.3 EGFP訊號在生物體器官表達的冷凍切片 25
3.5 CS-g-PEI/pCMV6-human insulin DNA的性質 26
3.5.1 功能DNA(functional DNA)的選定 26
3.5.1 CS-g-PEI/pCMV6-human insulin奈米微粒的粒子大小及界面電位 27
3.5.2 電子顯微鏡(TEM)圖像 27
3.5.3 CS-g-PEI/pCMV6-human insulin奈米微粒pH穩定性測試 28
3.6 CS-g-PEI/pCMV6-human insulin奈米微粒FRET測試 29
3.7 CS-g-PEI/pCMV6-human insulin奈米微粒轉運途徑的探討 31
3.8 CS-g-PEI/pCMV6-human insulin奈米微粒動物實驗 32
3.8.1 藥物效應動力學(pharmacodynamics, PD)測試 32
3.8.2 人類胰島素訊號在生物體器官表達的冷凍切片 32
3.8.3 小鼠全身對於血糖的利用情況 33
四、結論 35
五、參考文獻 36

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