博碩士論文 992203039 詳細資訊




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姓名 凃雅琪(Ya-chi Tu)  查詢紙本館藏   畢業系所 化學學系
論文名稱 研究新型奈米粒子載體結合核糖核酸干擾調控在細胞內蛋白之表現
(Designed Novel Nanocarriers for the Efficient Intracellular siRNA Delivery and Gene Silencing)
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摘要(中) 干擾性核醣核酸 (Ribonucleic acid Interference,RNAi),這個充滿潛力的應用,結合藥物設計將開啟醫療的新紀元。
  RNAi 作用場所在細胞膜內,所需克服的首要困難是必須使其具有透膜的特性。正確化學修飾提高 RNAi 的作用分子 siRNA (small interference RNA,siRNA),其穿過細胞膜低微可能性,保護 siRNA 抵禦酵素的降解的同時也保有釋放效率。相較於現今文獻中結構複雜的多功能載體,本實驗合成出了一個結構相對簡單的載體如 Scheme 1,由聚乙二醇 (Polyethylene glycol,PEG) 和 siRNA 為主體,兩者以具光裂解特性的連結體 (Photocleavable Linker,PL) 相連,其特性是照射 365 nm 波長的光會誘發其斷裂。
  此穿膜的載體設計的目的是期望以 PEG 提高載體疏水性及 siRNA 之穩定性;光照斷裂的連結體解決釋放上的調控問題,提高 RNAi 的效率。在沒有陽離子胜肽聚合物或是相反離子 (Counter Ion) 等高分子的協助下,此複合體依然自組裝形成結構完整的圓形粒子,實驗數據結果顯示,GFPsiRNA (Green Fluorescent Protein siRNA) 有機會被攜入進細胞內,抑制目標基因的趨勢。
摘要(英) Delivery of small interfering RNA (siRNA) has been one of the major hurdles for the application of RNA interference in therapeutics. The penetration of cell membrane for siRNA still exists the great difficulty. Thus, in order to overcome this challenge, we synthesized a complex with polyethylene glycol (PEG), photocleavable linker (PL), and thiolated siRNA for expecting success in siRNA penetration. Without incorporation of the cationic lipid or polymers with counter ions. PEG-PL-siRNA is still likely to form well-shaped particles. Interestingly, our data shows that those nanocarriers were effectively taken up by cells and then knocked down the expression of GFP gene.
關鍵字(中) ★ 奈米粒子載體
★ 短片段核糖核酸運輸
★ 綠色螢光蛋白
關鍵字(英) ★ nano carrier
★ GFP silencing
★ RNA interference
★ siRNA delivery
論文目次 謝誌 .......................................................................................................... I
中文摘要 ............................................................................................... III
英文摘要 ............................................................................................... IV
目錄 ........................................................................................................ V
Scheme ..................................................................................................VIII
Figure ........................................................................................................X
Table .........................................................................................................X
第一章 緒論 ..........................................................................................1
1-1 前言 .................................................................................................2
1-2 RNAi的發現 ...................................................................................3
1-3 RNAi 的機制 .................................................................................5
1-4 RNAi的應用與發展 .......................................................................8
1-4-1 RNAi的優勢 ........................................................................8
1-4-2 RNAi在醫療應用上的困難 ................................................8
1-4-3 迄今的發展 ..........................................................................9
1-4-4 RNAi在疾病治療上的應用................................................13
1-5 實驗動機及目的 ..........................................................................14
第二章 材料、合成及方法 ..................................................................16
2-1 載體設計 ...................................................................................... 17
2-1-1 聚乙二醇 PEG ...................................................................18
2-1-2 特殊修飾的 siRNA ...........................................................20
2-1-3 光誘發斷裂的連結體 PL ..................................................22
2-1-4 載體結構討論及光照斷裂過程 .......................................23
2-2 合成部分........................................................................................ 24
2-2-1 PL-PEG ...............................................................................24
2-2-2 PL-PEG 及 siRNA 的合成 .............................................24
2-3 實驗及鑑定儀器 ...................................................................27
2-3-1 高效能液相層析儀 (HPLC)...............................................27
2-3-2 可見光紫外光分光光譜儀 (UV/VIS Spectophotometer) 28
2-3-3 膠體電泳 (Gel Electrophoresis) ........................................28
2-3-4 動態光散射儀 (Dynamic Light Scattering) ......................39
2-3-5 表面電位分析儀 (Zeta Potential Analyzer) ......................30
2-3-6 穿透式電子顯微鏡 (Transmission Electron Microscopy) 30
2-3-7 細胞實驗 (In Vitro Test) ....................................................31
第三章 結果與討論 ....................................................................32
3-1 目標產物之結果與成效探討 siRNA21bp-PL-PEG5k ....................32
3-1-1 高效能液相層析 (HPLC) ..................................................33
3-1-2 電泳 (Electrolysis) .............................................................34
3-1-3 細胞實驗 (in vitro test) ......................................................35
3-1-4 動態散射光譜 (DLS) ........................................................36
3-1-5 穿透式電子顯微鏡 (TEM) ...............................................37
3-1-6 表面電位分析儀 (Zeta Potential Analyzer) ......................39
3-2 目標產物之構型與探討 ...............................................................39
3-3 不同光照連結體之差異探討 .......................................................39
3-3-1 動態散射光譜 .................................................................42
  3-3-2 穿透式電子顯微鏡之影像 ...............................................44
3-4 穿透細胞膜路徑之探討 ...............................................................46
第四章 結論 ........................................................................................48
第五章 參考文獻 ................................................................................50
第六章 附錄 ....................................................... ................................54
(附錄一) 含積分區域之各化合物層析圖
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指導教授 謝發坤(Fa-kuen Shieh) 審核日期 2012-8-20
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