新興糖基雙子型界面活性劑之結構以及其對基因轉染效率之影響

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姓名

黃信憲(Hsin-Hsien Huang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

新興糖基雙子型界面活性劑之結構以及其對基因轉染效率之影響
(Effect on Transfection Efficiency of the Structure of a Novel Sugar-based Gemini Surfactant)

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摘要(中)

基因治療是利用生物學方法將目的基因導入患者體內，使之達成目的基因產物，從而使疾病得到治療之療程，而高效的DNA轉染通常需要載體的協助。近年來，利用陽離子雙子型界面活性劑（通過將兩個界面活性劑分子與間隔物連接形成的二聚體狀界面活性劑）製成的奈米載體進行 DNA 轉染一直是科學家研究的熱門主題。然而，與病毒載體相比，這些非病毒載體的效率仍然很低。因此，許多科學家致力於研究影響轉染效率的因素，試圖做出改進。根據文獻，雙子型界面活性劑間隔基若擁有對 pH 的敏感的化學基團，可能提高其轉染效率。另一方面，糖基雙子型界面活性劑因其糖單元的可生物降解性和低細胞毒性而成為了非病毒載體的絕佳選擇之一。在這項研究中，我們合成了具有 N,N′-乙醯乙二胺間隔基的糖基雙子型界面活性劑，並探討其結構隨pH環境差異所產生的變化，進而研究結構對轉染效率影響。在這項研究中，我們發現糖基上的氫氧基與間隔基上的N,N′-乙醯乙二胺基都使整個雙子型界面活性劑的分子的結構，更容易因pH值的改變產生變化，且此界面活性劑能包覆DNA的能力極好，使其能夠達到非常高的轉染效率，因此，此項研究不管是在新材料的應用上，或是研究背後的機制上，必定能在細胞轉染的領域有所幫助。

摘要(英)

Gene therapy relies on DNA transfection to repair pathogenic genes, and efficient DNA transfection usually requires the assistance of carriers. DNA transfection by nanocarriers made of cationic gemini surfactants (a dimer-like surfactant formed by linking two surfactant molecules with a spacer) has been a hot topic for years; however, compared to virus-based carriers, the efficiency of these non-viral carriers remains low. Therefore, for the past decade, many scientists have looked into factors that determine the transfection efficiency in an attempt to make improvements. Earlier researches showed that certain chemical groups such as amines on the spacer of gemini surfactants endowed the surfactants with pH-sensitivity and might thereby improve their transfection efficiency. On the other hand, the sugar-based gemini surfactants have been a great choice for non-viral carriers due to the biodegradability and low cytotoxicity of their saccharide units. Accordingly, in this study, we synthesize a sugar-based gemini surfactant with a N,N’-acetylethylenediamine spacer, and explore the changes of its structure with the pH environment, and further study the effect of the structure on the transfection efficiency. In this study, we found that the hydroxyl group on the sugar group and the N,N′-acetylethylenediamine group on the spacer both make the molecular structure of the whole gemini surfactant more susceptible to the variety of pH value, and the ability of this gemini surfactant to form complexes with DNA is excellent, which enabling it to achieve a very high transfection efficiency. Therefore, this research will definitely be helpful for understanding the mechanism behind cell transfection and creating better biomaterial.

關鍵字(中)

★ 雙子型界面活性劑
★ 糖基雙子型界面活性劑
★ 基因轉染
★ 基因治療

關鍵字(英)

★ Gemini Surfactant
★ Sugar-base gemini surfactant
★ Gene transfection
★ Gene Therapy

論文目次

摘要 II
ABSTRACT III
致謝 IV
目錄 VI
圖目錄 VIII
表目錄 IX
第一章緒論 1
1-1 基因轉染 1
1-2 非病毒奈米載體 3
1-3 雙子型界面活性劑 5
1-4 糖基雙子型界面活性劑 7
1-5 酸鹼度敏感型雙子型界面活性劑 8
1-6 胞吞作用以及內體逃逸 10
1-7 研究目的與動機 13
第二章實驗方法 14
2-1 實驗材料 14
2-1-1 合成實驗藥品 14
2-1-2 實驗藥品 15
2-2 實驗儀器 17
2-3 雙子型界面活性劑合成 18
2-4 實驗方法 21
2-4-1 溶液製備 21
2-4-2 樣品製備 25
2-5 細胞實驗 27
2-5-1 HEK-293T繼代培養 27
2-5-2 轉染效率分析 28
2-5-3 載體包覆DNA之能力分析（電泳） 30
2-5-4 載體進入細胞之途徑分析（Flow cytometry） 31
2-6 分析儀器及測量方式介紹 32
2-6-1 核磁共振光譜（NMR） 32
2-6-2 動態光散射（DLS） 34
2-6-3 穿透式電子顯微鏡（TEM） 36
2-6-4 酵素免疫分析儀（ELISA reader） 38
2-6-5 DNA膠體電泳（Electrophoresis） 39
2-6-6 流式細胞分選儀（Flow Cytometer） 40
第三章結果與討論 42
3-1 合成結果 42
3-1-1 核磁共振光譜結果 42
3-1-2 界面活性劑性質分析 46
3-2 細胞實驗結果 53
3-2-1 載體包覆率實驗（電泳） 53
3-2-2 載體進入細胞之途徑分析 55
3-2-3 轉染效率分析 57
第四章結論 60
第五章文獻目錄與參考資料 62

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指導教授

陳儀帆(Yi-Fan Chen)

審核日期

2022-9-30

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