影響硬脂基化Indolicidin結構的因子及其基因傳輸效果的探討

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白旭閎(XU-HONG BAI) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

影響硬脂基化Indolicidin結構的因子及其基因傳輸效果的探討
(A study of factors that influence structure of stearylated Indolicidin and their effects on gene delivery)

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摘要(中)

為了提升C端硬脂基化Indolicidin (ILs) 的轉染效率，我們將輔助脂質二油酰磷脂酰乙醇胺(DOPE)與ILs以莫耳比為1:3混合 (ILs0.3L)，由先前實驗結果已知ILs0.3L之轉染效果高於ILs。為了瞭解其機制，我們進行了一連串的分析。由圓二色(CD)光譜得知，改質前的IL完全無α螺旋(α-helix)結構，ILs與ILs0.3L則於酸鹼環境皆有α-helix結構，表示IL在添加硬脂基後，有自組裝結構形成，因而導致二級結構改變。利用小角度X-ray散射(SAXS)、動態光散射(DLS)與穿透式電子顯微鏡(TEM)進行結構之構型及粒徑分析，在中性環境，ILs為柱狀微胞結構，ILs0.3L為單層囊泡結構，且ILs所形成的整體結構粒徑較大；而添加DNA後，發現ILs0.3L /DNA粒徑較ILs/DNA小，較易進入細胞。流式細胞儀實驗證實ILs0.3L與ILs皆以內吞作用將DNA帶入細胞，且ILs0.3L載體較ILs組多出巨胞飲作用進入細胞。在酸性環境下ILs0.3L/DNA則有結構重組以及零碎的結構散出的現象，且由肝素競爭實驗可知，因酸性環境時的結構不穩定，易與DNA分離，這應有助於基因藉由ILs0.3L攝入後的蛋白質表現。共軛焦顯微鏡實驗則顯示ILs0.3L與ILs皆有良好的內體逃脫能力。綜合上述結果，ILs0.3L因具有較小的粒徑、較多進入細胞的途徑、可由內體逃脫以及容易與DNA於胞內脫離等特性，使其傳輸能力高於ILs。

摘要(英)

To improve the transfection efficiency of C-terminal stearyl Indolicidin (ILs), a helper lipid, dioleoylphosphatidylethanolamine (DOPE), has been applied to mix with ILs at a molar ratio of 1:3 (ILs0.3L). Previous study indicates that ILs0.3L exhibits higher transfection efficiency than that of ILs. Therefore, a series of analyses were perfomed in this study to understand the promotion mechanism of helper lipids. The circular dichroism (CD) spectra showed that the structure of IL was completely random coiled, whereas ILs and ILs0.3L both exhibited partial α-helix properties. These results indicated that stearyl modiciation allow IL to self-assemble, which also led secondary structure formation. We used small angle X-ray scattering (SAXS), dynamic light scattering (DLS), and transmission electron microscope (TEM) to analyze self-assembled structures and their sizes. In a neutral environment, ILs and ILs0.3L formed cylindrical micelles and unilamellar vesicles, respectively. The overall structure of ILs0.3L was smaller than ILs. ILs0.3L/DNA was also smaller than ILs/DNA, suggesting that ILs0.3L/DNA may enter cells easier. The flow cytometry experiments showed that DNA delivered by ILs0.3L and ILs both through endocytosis. Interestingly, only ILs0.3L/DNA could enter cells through macropinocytosis. In an acidic environment, TEM results showed that the structure of ILs/DNA became compact, whereas ILs0.3L/DNA demonstrated structural reorganization and loose structures. Due to the unstability of ILs0.3L/DNA, the heparin competition experiment showed that acidic environment promoted DNA separated from ILs0.3L, which is essential for the protein expression of ingested genes. Confocal microscopy also showed that DNA delivered by ILs0.3L and ILs could both escape from endosome. Based on the abovementioned results, ILs0.3L owns smaller particle size, additional access to cells, and the capabilities of endosomal escape as well as intracellular DNA release, so its transfection efficiency is better than that of ILs.

關鍵字(中)

★ 胜肽
★ 載體
★ 脂質
★ 基因
★ 結構
★ 胞吞作用

關鍵字(英)

★ peptide
★ vector
★ lipid
★ gene
★ structure
★ endocytosis

論文目次

目錄
摘要 i
Abstract iii
致謝 iv
圖目錄 viii
表目錄 x
第一章緒論 1
1-1 研究背景 1
1-2 研究動機 2
1-3 實驗設計 3
第二章文獻回顧 4
2-1 基因治療 4
2-2 基因載體 6
2-3 自組裝結構(Self-assemble Structure) 7
2-3-1 小角X光散射(Small-Angle X-ray Scattering) 9
2-4 細胞穿膜胜肽(Cell Penetrating Peptides, CPPs) 12
2-4-1細胞穿膜胜肽之改質與應用 13
2-4-2 細胞穿膜胜肽與細胞膜間的作用機制 16
2-4-3 Indolicidin(IL) 18
2-5 脂質(Lipids) 19
2-5-1 脂質體轉染機制 20
2-5-2 輔助脂質(Helper lipids) 23
2-6 酸鹼度敏感載體(pH-sensitive vectors) 26
2-7 胜肽與脂質之複合載體 31
2-8 ILs與DOPE之複合物 34
第三章實驗藥品、儀器與方法 38
3-1 實驗材料 38
3-1-1 質體DNA 38
3-1-2 胜肽(Peptide) 39
3-1-3 細胞培養藥品 39
3-1-4 分析藥品 40
3-2 實驗儀器 43
3-3 實驗方法 45
3-3-1 溶液配置 45
3-3-2 胜肽/脂質(Peptide/Lipid)奈米粒子製備 48
3-3-3 質體DNA純化 49
3-3-4 HEK-293T細胞培養 49
3-3-5 載體及載體/DNA物性鑑定 53
3-3-6 載體進入細胞之途徑分析(Flow cytometer) 61
3-3-7 雷射共軛焦顯微鏡(Confocal) 63
3-3-8 肝素(Heparin)競爭實驗 64
3-4 SAXS數據分析 65
3-4-1 Cylinder model (SASVIEW；Version:5.0.5) 65
3-4-2 Core shell cylinder model (SASVIEW；Version:5.0.5) 65
3-4-3 Sphere model (SASVIEW；Version:5.0.5) 66
3-4-4 Unilamellar vesicle (form factor) /multilamellar vesicle (structure factor) (Matlab) 66
3-4-5 Vesicle model (SASVIEW；Version:5.0.5) 67
3-4-6 Multilayer vesicle model (SASVIEW；Version:5.0.5) 68
3-4-6 Asymmetric vesicle model (Origin) 68
第四章結果與討論 70
4-1 自組裝結構物性鑑定 70
4-1-1 二級結構(Secondary structure) 70
4-1-2 小角度散射(Small angle-X ray scattering) 72
4-1-3 表面電位 80
4-1-4 載體結構(TEM)與粒徑大小(DLS) 81
4-2 載體細胞攝取效果 93
4-2-1 流式細胞儀之途徑分析 (Flow cytometry) 93
4-2-2 雷射共軛焦顯微鏡 96
4-3 載體釋放DNA之能力 99
第五章結論 101
第六章參考文獻 103

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指導教授

胡威文(Wei-Wen Hu)

審核日期

2022-8-31

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