臺灣博碩士論文加值系統

富勒烯因為具有獨特的生物相容性和光電性質，因此在生物醫學領域中被廣泛的應用。另一方面，自然界中也有許多細胞膜蛋白，如細菌視紫質也同樣具有特殊的光電性質，高度表現的細菌視紫質（Highly expressible bacteriorhodopsin, HEBR）是一種新型的光敏蛋白，具有通過光遺傳學調節引發神經活動的潛力。在本研究中，我們將HEBR質體遞送至人類纖維母細胞中，並將其細胞培養在二維C60富勒烯自組裝奈米薄片上誘導其朝向神經分化。結果表明，上述方法結合綠光刺激（每天3次，一次持續3秒），可以在沒有任何神經誘導培養基的情況下促進人類纖維母細胞在7天內重新編程並分化成類神經細胞。將C60富勒烯作為培養基材，結合透過綠光刺激的HEBR進入細胞內，可提供新的光遺傳學平台，透過調節空間（C60奈米薄片）與時間（光照率）使人類纖維母細胞朝向類神經細胞發展，期待未來可應用於神經再生的組織工程。

Fullerenes have the unique biocompatibility and photoelectric properties and are candidate materials for biomedical applications. Several cell membrane proteins in nature such as bacteriorhodopsin also have photoelectric properties. Highly expressible bacteriorhodopsin (HEBR) is a novel light-sensitive opsin that has the potential to trigger neural activities through optogenetic modulation. In this study, we delivered HEBR plasmids to human fibroblasts and exposed the cells to C60 fullerene self-assembled two-dimensional nanosheets. Results showed that the above approach combined with light stimulation (3 second duration and three times per day) may promote the reprogramming and differentiation of human fibroblasts into neural-like cells in 7 days without any neural induction medium. The special photoelectric properties of fullerenes as culture substrates and transfected HEBR on cell membrane may provide a new optogenetic platform for regulating the location (C60 nanosheet) and time (frequency of light illumination) for human fibroblasts to become neural-like cells, and may be applied to improve neural regeneration in the future.

目錄
致謝 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 IX
表目錄 XI
第一章 文獻回顧 1
1.1. 碳材料簡介與生醫材料應用 1
1.1.1. 石墨烯 1
1.1.2. 奈米碳管 2
1.1.3. 富勒烯 3
1.2. 材料尺寸與生醫應用 5
1.3. 富勒烯自組裝之製備 6
1.3.1. 慢蒸餾法 7
1.3.2. 沈澱法和液-液介面沈澱法(LLIP) 7
1.4. 富勒烯自組裝之微結構 8
1.4.1. C60富勒烯奈米晶鬚 (C60 fullerene nanowhisker) 8
1.4.2. C60富勒烯奈米薄片(C60 fullerene nanosheet) 8
1.5. 光敏蛋白 9
1.6. 光敏蛋白結合奈米碳材之應用 10
1.7. 研究動機 11
第二章 研究方法 12
2.1. 研究架構圖 12
2.2. C60富勒烯奈米薄片之製備 14
2.3. C60富勒烯奈米薄片之物化性質分析 14
2.3.1. 掃描電子顯微鏡（Scanning electron microscopy, SEM） 14
2.3.2. 拉曼光譜儀 (Raman scattering) 15
2.3.3. X光繞射儀 (X-ray diffraction, XRD) 15
2.4. 覆蓋C60奈米薄片的基材 15
2.4.1. 覆蓋C60奈米薄片的基材之製備 15
2.4.2. C60奈米薄片基材之親水改質 16
2.4.3. C60奈米薄片基材之接觸角分析 16
2.5. 細胞培養 16
2.6. C60奈米薄片基材之細胞培養及增生率 17
2.7. HEBR質體轉染人類纖維母細胞 17
2.7.1. PolyFect轉染劑轉染HEBR質體之方法 17
2.7.2. 細胞轉染率 19
2.7.3. 細胞增生與存活率 19
2.7.4. 幹性基因表現 20
2.8. 光刺激和/或C60奈米薄片對HEBR轉染的纖維母細胞的影響 21
2.8.1. 神經相關基因表現 21
2.8.2. 神經相關蛋白免疫染色 22
2.8.3. 神經相關蛋白西方墨點法 22
2.9. 神經損傷斑馬魚模型之神經功能性實驗 23
2.10. 統計學分析 24
第三章 實驗結果 25
3.1. C60富勒烯奈米薄片物化性質之分析與鑑定 25
3.1.1. C60富勒烯奈米薄片之掃描式電子顯微鏡分析 25
3.1.2. C60富勒烯奈米薄片之拉曼光譜儀分析 25
3.1.3. C60富勒烯奈米薄片之廣角X-ray 繞射分析 25
3.2. C60奈米薄片玻璃基材之製備與表面改質 26
3.2.1. C60奈米薄片玻璃基材之製備 26
3.2.2. 以接觸角分析具Pluronic coating 的C60奈米薄片玻璃基材之表面 26
3.3. 在C60奈米薄片玻璃基材上的人類纖維母細胞之形態與增生率 27
3.3.1. 在C60奈米薄片玻璃基材上的人類纖維母細胞之形態 27
3.3.2在C60奈米薄片玻璃基材上的人類纖維母細胞之增生率 27
3.3.3. 在C60奈米薄片玻璃基材上的人類纖維母細胞之基因表現分析 28
3.4. HEBR質體轉染人類纖維母細胞 28
3.4.1. 細胞轉染後之HEBR質體之轉染率 28
3.4.2.細胞轉染後之存活率 29
3.4.3.細胞轉染後之增生率 29
3.4.4. 細胞轉染後之幹性相關基因表現分析 30
3.5. HEBR轉染的人類纖維母細胞在C60奈米薄片上的表現分析 30
3.5.1. 神經相關基因表現分析 30
3.5.2. 神經相關蛋白免液染色分析 31
3.5.3. 西方墨點法之神經蛋白表現分析 31
3.6. 斑馬魚神經損傷模型的功能測試 32
3.6.1. 成年斑馬魚神經損傷模型 32
3.6.2. 斑馬魚胚胎神經損傷模型 32
3.7. 在PU軟基材上覆蓋C60奈米薄片及其對人類纖維母細胞的影響 33
3.7.1. 在PU軟基材上覆蓋C60奈米薄片 33
3.7.2. 培養在C60奈米薄片PU基材上的人類纖維母細胞之形態 34
3.7.3. 在C60奈米薄片PU基材上的人類纖維母細胞之增生率 34
3.7.4. HEBR轉染的人類纖維母細胞在C60奈米薄片PU基材上的神經相關基因表現分析 34
第四章 討論 36
4.1. C60富勒烯奈米薄片物化分析 36
4.2. 表面親水性質對細胞貼附之影響 37
4.3. C60奈米薄片對細胞之影響 38
4.4. 轉染對細胞存活與增生率之影響 38
4.5. 幹性相關基因分析 39
4.6. 神經相關基因分析 39
4.7. 神經蛋白分析 40
4.8. 斑馬魚神經損傷模型的功能測試分析 41
4.9. 軟基材對於細胞之影響 41
4.9. 未來展望 42
第五章 結論 44
參考文獻 45
圖表 54
附錄一 藥品清單 68
附錄二 儀器清單 69

 
圖目錄
圖2.1. 研究架構圖 13
圖2.2. HEBR質體之蛋白質圖譜 18
圖2.3. 使用市售轉染試劑Polyfect作為遞送載體進行HEBR轉染 18
圖3.1. C60富勒烯奈米薄片的表面形貌 54
圖3.2. C60富勒烯奈米薄片的物化性質 55
圖3.3. 親水性C60奈米薄片基材的製備 56
圖3.4. 在C60奈米薄片玻璃基材上培養1天的人類纖維母細胞的形態 57
圖3.5. 在C60奈米薄片玻璃基材上培養的人成纖維細胞的增生數 57
圖3.6. C60奈米薄片作為細胞培養基材對纖維母細胞（無轉染HEBR質體）幹性的影響 58
圖3.7. 轉染並進一步培養1天後HEBR轉染的人類纖維母細胞的形態 59
圖3.8.（A）HEBR轉染的人類纖維母細胞的轉染效率（B）HEBR質體轉染對細胞存活率的影響（C）HEBR轉染之細胞增生的數量 59
圖3.9. 透過RT-PCR測定的HEBR轉染的纖維母細胞，轉染和培養5天後之幹性相關標記的基因表現 60
圖3.10. C60奈米薄片作為細胞培養基材對HEBR轉染的纖維母細胞的神經相關基因表現之影響 61
圖3.11. 將HEBR轉染的纖維母細胞接種在C60奈米薄片上7天後通過免疫螢光染色神經相關標記物的蛋白質表現 62
圖3.12.（A）通過西方墨點法確認的神經分化之能力（B）通過西方墨點法對蛋白質進行半定量分析 63
圖3.13. 植入通過在C60奈米薄片上培養的HEBR轉染的纖維母細胞，神經損傷的成年斑馬魚之功能恢復 64
圖3.14. 植入通過在C60奈米薄片上培養的HEBR轉染的纖維母細胞，神經損傷胚胎斑馬魚的功能恢復 65
圖3.15. 人類纖維母細胞在C60奈米薄片PU膜上的行為 66
圖3.16. 在C60奈米薄片PU膜上培養經HEBR質體轉染的纖維母細胞之基因表現 67 
表目錄
表2.1. 基因引子序列表 21

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