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研究生:陳令宜
研究生(外文):Emmeline Ling Yi Cheng
論文名稱:非病毒核酸載體應用於血管再狹窄預防之研究
論文名稱(外文):Non-viral gene delivery system for restenosis treatment
指導教授:劉澤英
指導教授(外文):Tse-Ying Liu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學工程學系
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:75
中文關鍵詞:抗血管再狹窄平滑肌細胞內皮細胞基因治療
外文關鍵詞:Anti-restenosisSmooth muscle cellEndothelial cellGene therapy
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血管狹窄發生後,進行介入性的血管形成術如汽球支架是廣為應用使血管回復到流通狀態的一種方法。但是,介入性手術後的高血管再狹窄機率是一個未解決的問題,使患者的晚期復原率仍然受限。而血管再狹窄的主要原因為血管中層的平滑肌細胞過度生長和血管內層的內皮細胞修復不及造成內膜增生。因此,本研究目的在設計一個抑制內膜增生的血管再狹窄預防方法。本研究使用雙親性幾丁聚醣衍生物、polyethylenimine高分子和核酸作為奈米載體的主要構成。載體攜帶的質體由適當的啟動子和功能性蛋白基因片段組成。我們首先用冷光酶試驗和西方點墨法最佳化載體組成比例和細胞轉染效果。再來用細胞存活率和TUNEL螢光看載體毒性和基因治療效果。最後用傷口癒合方法檢驗細胞遷移受治療的影響。我們期望這樣的治療方式能達到預防介入性治療後的血管再狹窄發生的目的。
The use of interventional vascular operation, such as stent placement with balloon catheter, is widely used as angioplasty therapy for vascular stenosis. However, the high rate of restenosis after operations is a drawback for long-term recovery of patients. The main reasons for this occurrence are smooth muscle cells over-growth and inefficient re-endothelialization. We used amphiphilic chitosan derivative, polyethylenimine and plasmid DNA to construct gene nano-vesicle. We confirmed the transfection efficiency and promoter activation efficiency in cells with luciferase assay and western blotting. Therapy inhibition effects were examined with cell viability assay and TUNEL assay. Last, cell migration effected by our therapy was also examined with wound healing assay. We expect our gene therapy approach can ultimately reduce the occurrence rate of vascular restenosis.
目錄
致謝 I
摘要 III
Abstract IV
目錄 V
圖目錄 VII
表目錄 VIII
第一章:緒論 1
第二章:文獻回顧 5
2.1血管再狹窄(restenosis) 5
2.1.1血管再狹窄機制探討 5
2.1.2平滑肌細胞於再狹窄處扮演的角色 6
2.1.3內皮細胞於再狹窄處扮演的角色 7
2.2血管再狹窄之現有治療方法 8
2.3血管再狹窄治療之遞送系統 12
2.3.1控制釋放系統 12
2.3.2選擇性抑制系統 12
2.3.3基因治療系統 13
2.4 PKCδ蛋白質機制探討 17
2.5選擇性開啟啟動子 18
2.6基因遞送系統 19
2.6.2非病毒載體 20
2.6.3高分子載體 21
2.6.4 Chitosan及其衍生物作為基因載體 23
2.6.4 PEI基因載體 23
第三章:材料與方法 25
3.1 材料 25
3.1.1 儀器 25
3.1.2 化學合成材料 26
3.1.3生物實驗材料 26
3.2 方法 28
3.2.1 載體材料合成與組裝 28
3.2.1.1 15N CHC合成 28
3.2.1.2 PEI-Au奈米粒子合成 28
3.2.1.3 胺基定量 29
3.2.1.4 奈米基因載體組裝 29
3.2.2 材料性質測試 29
3.2.2.1 穿透式顯微鏡 29
3.2.2.2 粒徑與表面電性測試 29
3.2.2.3 UV激發奈米金螢光 30
3.2.2.4 ICP-OES元素定量 30
3.2.2.5 NMR 30
3.2.2.6 FTIR 30
3.2.3 分子生物實驗 31
3.2.3.1 重組plasmid DNA 31
3.2.3.2 細菌培養 32
3.2.3.3 細菌放大DNA 32
3.2.3.4 DNA萃取 32
3.2.3.5 DNA Retardation Assay 32
3.2.4 細胞實驗 33
3.2.4.1 細胞培養 33
3.2.4.2 Plasmid DNA Transfection 33
3.2.4.3西方點墨法 33
3.2.4.4 Luciferase Assay 34
3.2.4.5 TUNEL Assay 34
3.2.4.6 Presto Blue Cell Viability Assay 34
3.2.4.7 Wound Healing Assay 34
3.2.4.8 Intracellular ROS Assay 35
3.2.5 統計方法 35
第四章:結果與討論 36
4.1 hEGR1細胞選擇性開啟效果驗證 36
4.2 phEGR1-PKCδ重組基因表達差異驗證 38
4.3 材料特性分析 40
4.4 PEI-Au/CHC/DNA奈米載體比例最佳化 43
4.5 PEI-Au/CHC/DNA奈米載體組成探討 47
4.6 PEI-Au/CHC/phEGR1-PKCδ誘導細胞凋亡效果 49
4.7 PEI-Au/CHC/phEGR1-PKCδ對細胞遷移的影響 59
4.8 Effectene/phEGR1-PKCδ誘導細胞凋亡效果 62
第五章:結論 66
第六章:參考資料 68

圖目錄
圖2-1. Mechanisms and consequences of inflammation after arterial injury 6
圖2-2. Proposed restenosis targets 6
圖2-3. Multiple mechanisms contribute to smooth muscle cell activation and growth 7
圖2-4. Causes and consequences of endothelial dysfunction after arterial balloon injury 8
圖2-5. PKCδ蛋白質構造 18
圖2-6. Schematic diagram of PKCδ-mediated apoptosis in vascular SMCs 18
圖2-7. Different injection routes of naked DNA and enhancement strategies 20
圖2-8. Schematic of the proton-sponge mechanism 22
圖4-1. hEGR1 promoter activity in smooth muscle cells and endothelial cells. 37
圖4-2. Schematic diagram of detailed plasmid constructs of phEGR1-Lucia and phEGR1-PKCδ. 39
圖4-3. The protein expression of PKCδ in smooth muscle cells and endothelial cells transfected with Effectene/hEGR1-PKCδ. 40
圖4-4. Characterizations of CHC and PEI-Au. 42
圖4-5. PEI-Au/CHC/DNA optimization 45
圖4-6. Comparison of PEI/CHC/DNA and PEI-Au/CHC/DNA. 46
圖4-7. Characterization of PEI/CHC/DNA and PEI-Au/CHC/DNA. 48
圖4-8. Cell viability of endothelial cells and smooth muscle cells treated with PEI-Au/CHC/DNA. 52
圖4-9. Nuclei morphology of cells treated with PEI-Au/CHC/DNA and LPS stimulation. 54
圖4-10. Nuclei morphology of cells treated with PEI-Au/CHC/DNA. 55
圖4-11. TUNEL assay tested on smooth muscle cells and endothelial cells with LPS stimulation. 56
圖4-12. TUNEL assay tested on smooth muscle cells and endothelial cells. 58
圖4-13. Wound healing assay 61
圖4-14. Cell viability of endothelial cells and smooth muscle cells treated with Effectene/DNA. 64
圖4-15. Schematic diagram demonstrating speculated roles of ER stress, nano-gold and PKCδ up-regulation. 65
圖5-1.奈米基因載體示意圖 66
圖5-2. 研究治療效果示意圖 67

表目錄
表2-1. Summary of Drug-Eluting Stent Clinical Data 10
表2-2. Pharmacological agents evaluated for their capacity to reduce the incidence of restenosis after balloon angioplasty 11
表2-3. Comparison of Principal Features of Restenotic Tissue after Bare-Metal and Drug-Eluting Stent Implantation. 11
表2-4. Summary of major studies using nonviral vectors for anti-restenosis gene therapy 15
表2-5. Summary of major studies using viral vectors for anti-restenosis gene therapy 16
表2-6. Design criteria for non-viral vectors 21
表3-1. 限制酶切割反應配置 31
表3-2. Ligation反應配置 31
表4-1. The amount of composition contained in the complex that added into one well on 96 well plate. 51


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