角膜表皮細胞受傷所引起的角膜基質細胞凋亡，是眼角膜疾病常見致病原因之一。本實驗利用先前研究出的胜肽奈米管，作為基因傳遞地載體，比較質體DNA在胜肽奈米管形成前與形成後加入，兩種不同混合質體DNA與胜肽奈米管的方式，與質體DNA的結合效果，並利用先前建立的角膜去除造成的角膜損傷模式，給予胜肽奈米管混合治療性質體DNA，評估胜肽奈米管混合質體DNA應用於治療角膜損傷的可行性。由原子力顯微鏡與雷射共軛焦顯微鏡可以觀察到所製出的胜肽奈米管長度約在2 ?慆以下，寬度約在200 nm以下。利用DNA會與胜肽奈米管上的酪胺酸(Tyr)產生交互作用，使用螢光光譜觀察胜肽奈米管與質體DNA的交互作用計算出質體DNA在胜肽奈米管形成後加入的結合常數為2.7x108 M-1，而質體DNA在胜肽奈米管形成前加入的結合常數為1.5x107 M-1。觀察兩種混合方式對DNase的安定性發現，則無明顯地差別。並以原子力顯微鏡與雷射共軛焦顯微鏡觀察到胜肽奈米管以及混合質體DNA的胜肽奈米管的型態。利用螢光顯微鏡觀察經由Thioflavin T 染色的胜肽奈米管投予至健康的裸鼠角膜上，可以觀察到胜肽奈米管穿透進入角膜表皮細胞與角膜基質細胞。在眼角膜去除之後的裸鼠眼角膜上，投予標定了Rhodamine的質體DNA與Thioflavin T標定的胜肽奈米管，利用螢光顯微鏡與雷射共軛焦顯微鏡發現胜肽奈米管與質體DNA亦能穿透進入角膜基質細胞。在傷害過後的裸鼠眼角膜上，給予混合胜肽奈米管和兩種不同抗細胞凋亡機轉的質體DNA(pCMV-Bcl-XL-eGFP和caspase 3 silencing shRNA)，並進行西方墨點法分析與Caspase 3 酵素活性試驗評估。

Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of peptide nanotubes(PNTs) as carrier of gene delivery between two mixing methods, plamid DNA added before PNTs forming or added after PNTs formed, was compared. Then a model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carrier on corneal injury. First, the PNTs were found under 2 ?慆 in length, 200 nm in width by atomic force microscope and confocal laser microscope system. Plasmid DNA were observed associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were added before and after PNT formation and were associated with tyrosine of PNTs with a binding constant of 2.7x108 M-1 and 1.5x107 M-1, respectively. The stability of plasmid DNA with PNTs against the DNase were found no difference between the two formulations. Using thioflavin T stained PNTs on the corneal eye drop delivery, PNTs were observed the distribution on epithelial and stroma region. After corneal debridement, the rhodamine labeled plasmid DNA and thioflavin T stained PNTs were also delivered and could be observed in the stroma of cornea. PNT complexed eyedrop delivery with two anti-apoptotic plasmids (pCMV-Bcl-xL-eGFP and caspase 3 silencing shRNA), were also evaluated by Western blot and caspase 3 activity.

目錄 1
圖表目錄 4
中文摘要 5
Abstract 6
壹、 緒論 7
一、基因傳遞 7
二、載體 9
三、角膜傷害與細胞凋亡 12
貳、 研究目的 17
參、 材料與研究方法 18
一、 實驗試藥 18
二、 儀器與耗材 19
三、 溶液、試劑製備 20
1. Agar plate 20
2. Alkaline Lysis Solution I 21
3. Alkaline Lysis Solution II 21
4. Alkaline Lysis Solution III 21
5. Ethidium Bromide (10mg/ml) 21
6. NaOH (10N) 21
7. 20% SDS 21
8. Tris-HCl (1M; pH 8.8, 7.4, 6.8) 22
9. EDTA (0.5M; pH 8.0) 22
10. 2X SDS gel-loading buffer 22
11. 10X Tris-EDTA buffer (10X TE buffer) 22
12. 1X Tris-EDTA buffer (1X TE buffer) 23
13. 5X Tris-glycine buffer 23
14. 1X Tris-glycine buffer 23
15. 50X TAE buffer 23
16. 0.5X TAE buffer 23
17. Transfer Buffer 23
18. Phosphate Buffer Saline (PBS) 24
19. LB medium 24
20. 10X Tris buffered saline (10X TBS) 24
21. TBST 24
22. Stripping Buffer 24
四、 實驗動物 25
五、 研究方法 25
1. 製備胜肽奈米管 25
2. 製備質體DNA 25
3. TM-Rhodamine標記質體DNA 27
4. 原子力顯微鏡 28
5. 螢光光譜偵測 28
6. 質體DNA混合胜肽奈米管對DNase之安定性試驗 28
7. Zeta potential測量 29
8. Thioflavin T標記胜肽奈米管 29
9. 裸鼠角膜剝除傷害模式 29
10. 活體動物眼藥水投予 30
11. 西方墨點法分析 30
12. Caspase 3 活性分析 31
13. 組織切片分析 31
肆、 實驗結果與討論 32
一、 胜肽奈米管之物化性質 32
1. 胜肽奈米管於顯微鏡下觀察 32
2. Zeta potential測量 34
3. 螢光光譜分析 35
二、 胜肽奈米管混合質體DNA於DNA分解酵素之安定性分析 38
三、 裸鼠投予胜肽奈米管眼藥水之觀察 39
四、 裸鼠角膜傷害與投藥後觀察 41
1. 裸鼠角膜傷害模式 41
2. 螢光標定之質體DNA與胜肽奈米管於顯微鏡觀察 41
3. 西方墨點法分析 44
4. Caspase 3 酵素活性分析 45
伍、 結論 46
陸、 參考文獻 47

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