博碩士論文 105324024 詳細資訊




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姓名 李韋成(Wei-Cheng Li)  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 Indolicidin之色胺酸殘基對於轉染效率的影響
(The Effect of Tryptophan Residues on The Transfection Efficiency of Indolicidin)
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摘要(中) 在先前的研究當中,我們將Indolicidin(IL) 胜?的N端與C端分別接上半胱胺酸,並分別命名為ILC與CIL。由分子動態模擬發現接枝於聚乙烯亞胺(PEI)的IL是由色胺酸將其疏水片段帶入細胞膜內,來達到基因輸送的效果。為了探討色胺酸對轉染的重要性,因此本研究將ILC序列中第四與第八個色胺酸以及CIL序列中第八與第九個色胺酸改成親水性的甘胺酸,分別命名為ILC48與CIL89,將其以共價接枝或直接摻混結合PEI,以進行質體DNA輸送。由溶血性實驗證實ILC與CIL有強烈的溶血性,反觀改質的ILC48與CIL89則有效降低溶血現象,此趨勢與由MTT assay結果一致,證實減少色胺酸可有助於生物安全性。轉染實驗結果顯示,在接枝的情況下,PEI-ILC及PEI-CIL均可有效地促進轉染,但是PEI-ILC48和PEI-CIL89兩組載體幾乎沒有轉染效率。在直接摻混的實驗方面,由於胜?可藉由其半胱胺酸形成雙硫鍵而成為二元體,而結果顯示,此二元體形式的胜?更能促進轉染效率,其中ILC48與CIL89均有顯著地提昇PEI的轉染效果。除了因為其色胺酸減少所降低的毒性,二元體形式的胜?帶電量較高且分子較長,推測能有助於其與DNA複合的比例,且可提高基因在胞內與載體分離。透過本研究我們了解Indolicidin序列上的色胺酸對於基因輸送的重要性,並有助於設計更安全與高效率的非病毒載體。
摘要(英) In the previous study, Indolicidin (IL) was added a cystein to its N- and C- terminus, which were denoted as CIL and ILC, respectively. Using molecular dynamics simulations, we found that the grafted IL inserted into the cell membrane mainly related to their tryptophan residues. In order to validate these simulation results, the fourth and eighth tryptophans in the ILC sequence and the eighth and ninth tryptophans in the CIL sequence were replaced with glycine, which were denoted as ILC48 and CIL89, respectively. The hemolysis assay demonstrated that ILC and CIL were highly hemolytic. In contrast, hemolysis caused by ILC48 or CIL89 was low. This trend was consistent to the results of MTT assay, suggesting the reduction of tryptophan should be beneficial to biosaftey. For gene delivery purpose, these peptides were combined with PEI either as conjugates or as mixture to deliver plasmid DNA. In the conjugation form, different from PEI-ILC and PEI-CIL which demonstrated good transfection efficiency, PEI-ILC48 and PEI-CIL89 were unable to transfect cell. Then, we mixed peptides and PEI with DNA as ternary nanocomplexes for transfection. These cysteine-containing peptides can form as dimers through disulfide bond formation. Compared to the monomeric form, dimeric peptides demonstrated superior transfection efficiency. Especially, ILC48 and CIL89 were capable of promoting PEI-mediated gene delivery. We deduced that the dimeric peptide owned more positive charges and larger molecular weights, which increased their complexing ratio to DNA. In addition, their cleavage also facilitated DNA release from peptides intracellularly. Through this comprehensive study, we can determine the importance of tryptophan in peptide for gene delivery. And these results may provide useful information to design a safe and efficient non-viral vector.
關鍵字(中) ★ 聚乙烯亞胺
★ 胜?
★ 二元體
★ 基因傳遞
★ 生物偶聯
★ 色氨酸
關鍵字(英) ★ tryptophan
★ polyethylenimine
★ peptides
★ dimer
★ gene delivery
★ bioconjugation
論文目次 目錄
摘要 I
Abstract III
致謝 IV
目錄 V
圖目錄 IX
表目錄 XI
第一章 緒論 1
1-1背景 1
1-2實驗動機 4
第二章 文獻回顧 5
2-1基因療法 5
2-2基因載體 6
2-2-1 脂質體複合物 7
2-2-2 正電高分子及其高分子複合物 8
2-3穿膜胜? 12
2-3-1 穿膜胜?的種類與特性 12
2-3-2 Indolicidin 14
2-4利用穿膜胜?與聚乙烯亞胺進行基因輸送 16
2-4-1胜?接枝於聚乙烯亞胺 16
2-4-2三成分摻混對基因的輸送 17
2-5 穿膜胜?的擾膜機制 19
2-6 以二元體形式應用於藥物輸送 22
第三章 實驗材料與方法 24
3-1 試藥與原料 24
3-1-1質體DNA 24
3-1-2細胞 25
3-1-3穿膜胜?(Cell-penetrating peptides) 25
3-1-4藥品 26
3-1-5細胞培養用藥 28
3-2 儀器 28
3-3 試藥配製 29
3-4 質體DNA純化 32
3-5 NIH-3T3細胞培養 33
3-6 PEI結合不同種類CPP對轉染效率影響 36
3-6-1細胞轉染 36
3-6-2 ONPG轉染效率分析 41
3-6-3 MTT分析 43
3-7 奈米粒子製備及物理化學性質鑑定 44
3-7-1雷射粒徑分佈儀(dynamic light scattering, DLS)分析 44
3-7-2包覆率分析 44
3-8 Indolicidin之溶血活性 46
3-9 蛋白質膠體電泳(SDS-PAGE)分析 47
3-10載體緩衝能力分析 50
第四章 結果與討論 51
4-1胜?接枝PEI應用於藥物輸送 52
4-1-1 PEI衍生物之緩衝能力 52
4-1-2奈米粒子物性鑑定 54
4-1-3粒子包覆率 58
4-1-4溶血性實驗 62
4-1-5 MTT 測試對細胞活性分析 63
4-1-6 PEI結合穿膜胜?對轉染效率之影響 65
4-2胜?與PEI簡單複合應用於藥物輸送 67
4-2-1 SDS-PAGE分析 67
4-2-2奈米粒子物性鑑定 69
4-2-3粒子包覆率 71
4-2-4 Peptide溶血性實驗 74
4-2-5 MTT 測試對細胞活性分析 75
4-2-6 PEI結合穿膜胜?對轉染效率之影響 77
第五章 結論 80
參考文獻 82
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指導教授 胡威文(Wei-Wen Hu) 審核日期 2018-8-23
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