在組織工程上，生長因子和細胞為神經損傷修復中重要的影響因子，在醫療策略上給予外源性的生長因子，可保護損傷的神經組織並促進再生，來改善神經傳導的功能。鹼性纖維母細胞生長因子(basic fibroblast growth factor, bFGF)可有效地促進血管新生與神經細胞的生長，然而bFGF半生期短而且容易被酵素所降解，因此本次研究主要利用天然聚合物-幾丁聚醣(Chitosan, C)與帶有硫酸基團的褐藻醣膠(Fucoidan, F)製作成生物可降解的載體，攜帶bFGF以及維持bFGF之活性，並藉由奈米粒控制釋放bFGF來修復受損的神經組織，幫助神經細胞的再生。實驗結果顯示：幾丁聚醣與褐藻醣膠藉由靜電作用力可形成穩定之奈米粒，粒徑範圍在60~600 nm之間；此外，褐藻醣膠的濃度影響奈米粒的性質。以0.5%(w/v)褐藻醣膠所製成之幾丁聚醣/褐藻醣膠奈米粒，可有效保護bFGF使其在溫度及酵素作用下仍保有活性，並控制釋放bFGF。細胞實驗結果亦顯示：由幾丁聚醣/褐藻醣膠奈米粒釋放之bFGF有助於PC12細胞增生，並可在分化初期加速PC12細胞進行分化的表現。此幾丁聚醣/褐藻醣膠奈米粒為一個有效且具有功能性的奈米載體，未來將以動物實驗來模擬臨床人類神經損傷治療的可行性。

In tissue engineering, growth factors and cells are important elements for repairing nerve injuries. Supplemented with exogenous growth factors is an effective medical strategy for protecting nerve injury and promoting nerve regeneration. It has been demonstrated that basic fibroblast growth factors have great effect on angiogenesis and neuronal cell survival; however, bFGF has two shortages: short half-life and easily degraded by enzymes. Therefore, the aim of this study is to design a biodegradable chitosan/fucoidan (C/F) nanoparticle to carry bFGFs and to maintain their activities. Furthermore, apply the bFGFs released from nanoparticles to promote nerve regeneration. According to our experimental results, chitosan and fucoidan formed stable nanoparticles by electrostatic interactions with nanoparticles size at 60~600 nm. The concentration of fucoidan affected the characteristics of C/F nanoparticles. At 0.5% (w/v) fucoidan solution, the prepared C/F nanoparticles not only protected bFGF from deactivation by temperature and enzymes but also controlled the release of bFGFs. By in vitro cell studies, there was no cytotoxicity observed on PC12 cells. The differentiation of PC12 cells were promoted by released bFGF in the early stage. Briefly, C/F nanoparticles are potential carriers for delivery bFGFs. The effect of bFGFs released from C/F nanoparticles on axonal elongation and animal experiments will be studied as a preclinical trial before clinical application in the near future.

目錄
謝誌 I
摘要 II
Abstract III
圖目錄 VIII
表目錄 IX
第一章、前言 1
第二章、文獻回顧 3
2.1 神經系統 3
2.2 神經修復 4
2.3 神經再生之技術 5
2.4 鹼性纖維母細胞生長因子（bFGF） 6
2.5 奈米技術 7
2.5-1 奈米技術 7
2.5-2奈米粒藥物傳輸系統 7
2.5-3 控制釋放 8
2.6 幾丁聚醣 8
2.6-1 幾丁聚醣基本結構 8
2.6-2 幾丁聚醣之生理活性 9
2.6-3幾丁聚醣於生物醫學材料上之應用 9
2.7 褐藻醣膠 12
2.7-1 褐藻醣膠基本結構 12
2.7-2 褐藻醣膠之生理活性 12
2.7-3 褐藻醣膠於生物醫學材料上之應用 13
2.8 腎上腺髓質嗜鉻細胞瘤(Rat adrenal phromecytoma) 14
第三章、實驗動機與目的 15
第四章、實驗架構 16
第五章、實驗材料與方法 17
5.1 實驗藥品 17
5.2 實驗儀器 20
5.3 實驗溶液配製 21
5.4 實驗步驟 24
5.4-1 幾丁聚醣分子量降解 24
5.4-2 褐藻醣膠硫酸化之定量法 24
5.4-3 幾丁聚醣/褐藻醣膠奈米粒製備 25
5.4-4 材料物理性質測試 26
5.4-5 酵素結合免疫吸附法（ELISA）測量bFGF控制釋放量 28
5.4-6 奈米粒對bFGF之間之交互作用影響 29
5.4-7 體外細胞實驗 30
5.4-8 細胞影像觀察 34
第六章、結果與討論 35
6.1奈米粒之材料性質分析 35
6.2不同製備方法之比較 36
6.3不同組成之奈米粒對bFGF之間之交互作用影響 36
6.3-1 奈米粒數量分析 36
6.3-2 粒徑大小與界達電位分析 37
6.3-3 保護試驗-熱與酵素處理 38
6.3-4 包覆率和控制釋放結果 39
6.3-5 奈米粒對bFGF之結合試驗 40
6.4 體外細胞實驗 40
6.4-1 奈米粒之細胞毒性結果 40
6.4-2 奈米粒控制釋放bFGF對細胞生長之影響 40
6.4-3 不同濃度之褐藻醣膠對細胞活性表現 41
6.4-4 奈米粒對分化後細胞之生長、分化影響 42
第七章、結論 45
第八章、未來展望 47
第九章、.參考文獻 48
第十章、圖表 56


圖目錄
圖 1. 實驗架構圖 16
圖 2. 週邊神經束的解剖構造 56
圖 3. 週邊神經修復示意圖 57
圖 4. 中樞神經損傷示意圖 58
圖 5. 幾丁聚醣化學結構 59
圖 6. 褐藻醣膠之化學結構 60
圖 7. 方法1 之製作示意圖 61
圖 8. 方法2之製作示意圖 62
圖 9. 體外細胞實驗之架構 63
圖 10. PC12細胞分化之定義 64
圖 11. 幾丁聚醣、褐藻醣膠和幾丁聚醣/褐藻醣膠奈米粒FT-IR圖譜 65
圖 12. 幾丁聚醣/褐藻醣膠奈米粒(C1F1)之粒徑分佈圖與TEM圖 66
圖 13. 方法1與方法2之包覆率結果 67
圖 14. 方法1與方法2之控制釋放結果 68
圖 15. 不同組成之幾丁聚醣/褐藻醣膠奈米粒。(1)C1F10；(2)C1F5；(3)C1F1；(4)C5F1；(5)C10F1；(A)未離心；(B)離心後。 69
圖 16. 利用透光率分析不同條件下之奈米粒數量 70
圖 17. 幾丁聚醣/褐藻醣膠奈米粒穩定性實驗。（A）C1F10；（B）C1F5；（C）C1F1；（D）C5F1；（E）C10F1 71
圖 18. 幾丁聚醣/褐藻醣膠奈米粒經熱處理後對BFGF之保護試驗 72
圖 19. 幾丁聚醣/褐藻醣膠奈米粒經酵素處理後對BFGF之保護試驗 73
圖 20. 不同條件下之奈米粒之包覆率 74
圖 21. 不同條件下之奈米粒之控制釋放 75
圖 22. 幾丁聚醣/褐藻醣膠奈米粒對BFGF之結合試驗. 76
圖 23. 幾丁聚醣/褐藻醣膠奈米粒(無BFGF)之毒性實驗 77
圖 24. 幾丁聚醣/褐藻醣膠奈米粒(有BFGF)對PC12細胞之存活率表現 78
圖 25. 不同濃度之褐藻醣膠對細胞抑制生長之表現 79
圖 26. 奈米粒控制釋放BFGF對分化後細胞之生長表現 80
圖 27. 奈米粒控制釋放BFGF對分化後細胞之分化表現 81
圖 28. 細胞型態圖 82

表目錄
表 1. 不同濃度之幾丁聚醣和褐藻醣膠所組成的幾丁聚醣/褐藻醣膠奈米粒 83
表 2. 奈米粒對分化後細胞之生長、分化實驗條件 84
表 3. 不同組成奈米粒之粒徑大小與界達電位 85

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