新穎基因/ 藥物輸送系統之開發與評估__臺灣博碩士論文知識加 ...

2024-09-21

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而本研究主要是在開發一系列新穎的基因傳遞系統以及藥物釋控平台， ... 與陽離子型聚胺基甲酸酯之掺合技術來增加DNA傳遞效率及降低細胞毒性之研究、（3）新穎陽離子型 ... 資料載入處理中... 跳到主要內容臺灣博碩士論文加值系統 ::: 網站導覽| 首頁| 關於本站| 聯絡我們| 國圖首頁| 常見問題| 操作說明 English |FB專頁 |Mobile 免費會員登入| 註冊功能切換導覽列 (165.22.106.144)您好！臺灣時間：2022/09/0501:03 字體大小： ::: 詳目顯示 recordfocus 第1筆/ 共1筆 /1頁論文基本資料摘要外文摘要目次參考文獻電子全文紙本論文 QRCode 本論文永久網址: 複製永久網址Twitter研究生:洪維志研究生(外文):Hung,Weichih論文名稱:新穎基因/藥物輸送系統之開發與評估論文名稱(外文):Constructionandevaluationofnovelgene/drugdeliverysystems指導教授:程中玉指導教授(外文):Cherng,JongYuh口試委員:朱延和、洪伯誠、程中玉、蕭明達、施美份口試委員(外文):Chu,Yen-Ho、Hong,Bor-Cherng、Cherng,JongYuh、Shau,MinDa、Shih,MeiFen口試日期:2011-05-10學位類別:博士校院名稱:國立中正大學系所名稱:化學暨生物化學研究所學門:自然科學學門學類:化學學類論文種類:學術論文論文出版年:2011畢業學年度:99語文別:中文論文頁數:300中文關鍵詞:醫學材料、生物可降解性、轉染、藥物釋控、細胞毒性、超音波外文關鍵詞:biomaterial、biodegradable、transfection、drugcontrolrelease、cytotoxicity、ultrasound相關次數: 被引用:0點閱:314評分:下載:18書目收藏:0 隨著科技的進步、生活品質提升，當前人類最重視的是醫療科技方面的突破性發展。

奈米技術的衝擊將醫藥及生技推向更高的水平。

而近幾年發展中，生物相容性的醫學材料（Biomaterials）已被廣泛地應用於臨床治療，將這些低毒性的材料發展為於藥物或治療基因的載體已逐漸受到重視而嶄露頭角。

而本研究主要是在開發一系列新穎的基因傳遞系統以及藥物釋控平台，並藉由一些生物可降解性高分子或是適當的物理性刺激來達到基因傳遞以及藥物釋控上。

本研究將分六部份分別陳述（1）陽離子型聚氨基甲酸酯之合成及其結構上胺基對DNA轉染之影響、（2）利用Polyethylenimine與陽離子型聚胺基甲酸酯之掺合技術來增加DNA傳遞效率及降低細胞毒性之研究、（3）新穎陽離子型聚胺基甲酸酯接枝PEI之合成與複合式基因傳遞系統之開發、（4）合成與鑑定一系列新型生物可降解性聚胺基亞胺之基因載體、（5）利用超音波基因傳遞技術改良基因傳遞系統之研究、與（6）新穎奈米型藥物釋控材料PEG-dsDNA/Lipofectamine之開發及細胞內藥物釋控之研究。

Withtheprogressofsciencedevelopmentandimprovementoflifequality,abreakthroughinmedicaltechnologyhasbeenurgentlyrequired.Theimpactofnano-techniqueinfluencesmodernmedicineandtechnologytowardtheirlimits.Biomaterialshavebeenwidelyinclinicalapplicationspresently.Theselowcytotoxicmaterialshaveattractedmanyinterestsandattentionindevelopmentofdrugcarriersorgenevectors.Themaingoalofthisstudyistoestablishaseriesofgenedeliverysystemsandaplatformofdrugcontrolrelease,andtousenewbiodegradablepolymersorsuitableenhancementingene/drugdelivery.Thisworkisdividedintosixtopics,including(1)thesynthesisofcationicpolyurethanestostudytheeffectofaminesandstructuresontheirDNAtransfectionpotential,(2)blendingofpolyethyleniminewithacationicpolyurethanegreatlyenhancesbothDNAdeliveryefficacyandreducestheoverallcytotoxicity,(3)synthesisofnovelcationicpolyurethanegraftedPEIanddevelopmentinmulti-genedeliverysystem,(4)synthesisandcharacterizationsofaseriesofnovelbiodegradablepolyaminoimineasgenecarriers,(5)improvementofagenedeliverysystembyultrasonictransfectiontechnology,and(6)establishmentofnovelnanoscaledrugcarriersfromPEG-dsDNA/Lipofectamineandtheirapplicationsinintracellulardrugdelivery. 目錄中文摘要...............................................................................................................i英文摘要..............................................................................................................ii謝誌.....................................................................................................................iv要目………………………………………………………………………….…vi目錄.....................................................................................................................xi圖表目錄.........................................................................................................xxix第一部份緒論....................................................................................................1第一章生醫材料................................................................................................11-1生醫材料的發展........................................................................................1第二章基因治療................................................................................................32-1基因治療的起源........................................................................................32-2基因治療的原理........................................................................................52-3基因傳遞系統............................................................................................62-3-1物理性基因傳遞方式..........................................................................72-3-1-1電穿孔的方式................................................................................72-3-1-2超音波............................................................................................72-3-1-3注射與微注射................................................................................82-3-1-4基因槍............................................................................................82-3-2化學式基因傳遞方式............................................................................92-3-2-1磷酸鈣－DNA共沉澱法...............................................................92-3-3基因載體傳遞方式..............................................................................92-3-3-1病毒性載體....................................................................................92-3-3-1-1反轉錄病毒............................................................................102-3-3-1-2腺病毒....................................................................................112-3-3-1-3腺病毒相關病毒......................................................................122-3-3-2非病毒性載體..............................................................................122-3-3-2-1微脂粒載體............................................................................132-3-3-2-2陽離子型高分子載體............................................................132-4陽離子型高分子載體的轉染機制..........................................................152-5基因轉殖的機制與障礙因素..................................................................182-5-1標的細胞之再吸收............................................................................182-5-2內質體之釋放....................................................................................182-5-3在細胞質中安定性與細胞核的輸入................................................19第二部份陽離子型聚氨基甲酸酯之合成及其結構上胺基對DNA轉染之影響......................................................................................................20第一章研究背景與目的..................................................................................21第二章研究動機..............................................................................................23第三章實驗流程..............................................................................................263-1實驗藥品..................................................................................................273-2實驗儀器與裝置......................................................................................283-3名詞對照..................................................................................................293-4實驗步驟..................................................................................................293-4-1聚胺基甲酸酯主鏈（PU）之合成.........................................................293-4-2不同型態側鏈的PU之合成................................................................303-4-2-1一級胺型態側鏈之PU（PU1）......................................................303-4-2-2二級胺型態側鏈之PU（PU2）......................................................303-4-2-3三級胺型態側鏈之PU（PU3）......................................................303-4-3含環氧丙醇的PU之合成..................................................................313-4-4不同型態側鏈的PU-環氧丙醇之合成...............................................313-4-4-1一級胺型態側鏈之PU-glycido（PUg1）......................................313-4-4-2二級胺型態側鏈之PU-glycido（PUg2）......................................313-4-4-3三級胺型態側鏈之PU-glycidol（PUg3）.....................................323-4-5材料合成之鑑定................................................................................323-4-6PlasmidDNA之製備...........................................................................323-4-7PU(1-3)/DNA複合體與PUg(1-3)/DNA複合體之製備..................333-4-8Gelelectrophoresis分析......................................................................333-4-8-1restrictionenzyme的保護分析.....................................................333-4-8-2DNaseI的保護分析......................................................................343-4-9粒徑分析..............................................................................................343-4-10高分子之酸鹼滴定（緩衝能力）分析................................................343-4-11PU水解之Gelelectrophoresis分析..................................................353-4-12細胞培養..........................................................................................353-4-13細胞毒性測試（XTTassay）.............................................................363-4-14polymer/DNA複合體之體外基因轉染試驗...................................36第四章結果與討論..........................................................................................374-1聚胺基甲酸酯主鏈（PU）之合成...............................................................374-2不同型態側鏈的PU之合成......................................................................384-3不同型態側鏈的PU-環氧丙醇之合成...................................................434-4Gelelectrophoresis分析............................................................................464-5restrictionenzyme與DNaseI的保護分析...............................................484-6粒徑分析..................................................................................................504-7高分子之酸鹼滴定（緩衝能力）分析........................................................524-8PU水解之Gelelectrophoresis分析..........................................................534-9細胞毒性測試（XTTassay）......................................................................544-10PU/DNA複合體之體外基因轉染試驗.................................................56第五章結論......................................................................................................58第三部份利用Polyethylenimine與陽離子型聚胺基甲酸酯之掺合技術來增加DNA傳遞效率及降低細胞毒性之研究.....................................60第一章研究背景與目的..................................................................................61第二章研究動機..............................................................................................63第三章實驗流程..............................................................................................653-1實驗藥品..................................................................................................663-2實驗儀器與裝置......................................................................................663-3名詞對照..................................................................................................673-4實驗步驟..................................................................................................683-4-1聚胺基甲酸酯主鏈（PU）之合成.........................................................683-4-2含環氧丙醇的PU之合成..................................................................683-4-3三級胺型態側鏈之PU-glycidol（PUg3）...........................................693-4-4材料合成之鑑定................................................................................693-4-5PlasmidDNA之製備...........................................................................693-4-6PUg3/PEI/DNA複合體之製備........................................................703-4-7Gelelectrophoresis分析......................................................................703-4-8粒徑分析..............................................................................................713-4-9細胞培養............................................................................................713-4-10細胞毒性測試（XTTassay）.............................................................713-4-11PAI/DNA複合體之體外基因轉染試驗..........................................72第四章結果與討論..........................................................................................734-1聚胺基甲酸酯主鏈（PU）之合成...............................................................734-2PUg3之合成..............................................................................................734-3Gelelectrophoresis分析............................................................................744-4粒徑分析..................................................................................................764-5細胞毒性測試（XTTassay）......................................................................774-6PUg3/PEI/DNA複合體之體外基因轉染試驗......................................78第五章結論......................................................................................................80第四部份合成與鑑定一系列新型生物可降解性聚胺基亞胺之基因載體....................................................................................................81第一章研究背景與目的..................................................................................82第二章研究動機..............................................................................................87第三章實驗流程..............................................................................................903-1實驗藥品..................................................................................................923-2實驗儀器與裝置......................................................................................933-3名詞對照..................................................................................................943-4實驗步驟..................................................................................................943-4-1聚胺基甲酸酯主鏈（PU）之合成.........................................................943-4-2PU-PEI600之合成.................................................................................953-4-3材料合成之鑑定................................................................................953-4-4PlasmidDNA之製備..........................................................................963-4-5PU-PEI600複合體之製備.....................................................................963-4-6Gelelectrophoresis分析......................................................................963-4-7粒徑分析..............................................................................................963-4-8高分子之酸鹼滴定（緩衝能力）分析..................................................973-4-9細胞培養............................................................................................973-4-10細胞毒性測試（XTTassay）.............................................................973-4-11PU-PEI600/DNA複合體之體外基因轉染試驗................................983-4-12PU-PEI之穩定性測試......................................................................983-4-13PU-PEI600-maleimide之合成............................................................983-4-14材料合成之鑑定..............................................................................993-4-15PlasmidDNA（GFP、RFP）之製備....................................................993-4-16PU-PEI600-maleimide/DNA複合體之製備....................................993-4-17Gelelectrophoresis分析....................................................................993-4-18粒徑分析..........................................................................................993-4-19PU-PEI-maleimide/DNA複合體之體外基因轉染試驗...............1003-4-20複合式基因複合體之粒徑分析....................................................1003-4-21複合式基因複合體之細胞毒性分析..............................................1003-4-22diSH化合物之細胞毒性分析（XTTassay）...................................1003-4-23複合式DNA體外基因轉染試驗...................................................100第四章結果與討論........................................................................................1024-1聚胺基甲酸酯主鏈（PU）之合成.............................................................1024-2PU-PEI600之合成.....................................................................................1024-3Gelelectrophoresis分析..........................................................................1044-4粒徑分析................................................................................................1054-5PU-PEI600之酸鹼滴定（緩衝能力）分析.................................................1064-6細胞毒性測試（XTTassay）....................................................................1074-7PU-PEI600/DNA複合體之體外基因轉染試驗......................................1084-8PU-PEI600之穩定性測試.........................................................................1104-9PU-PEI600-maleimide之合成..................................................................1114-10Gelelectrophoresis分析........................................................................1134-11粒徑分析..............................................................................................1144-12PU-PEI-maleimide/DNA複合體之體外基因轉染試驗.....................1154-13複合式基因複合體之粒徑分析..........................................................1164-14複合式基因複合體之細胞毒性分析（XTTassay）.............................1174-15diSH化合物之細胞毒性分析（XTTassay）.........................................1184-16複合式DNA體外基因轉染試驗.........................................................119第五章結論....................................................................................................126第五部份合成與鑑定一系列新型生物可降解性陽離子高分子：聚胺基亞胺之基因載體開發............................................................................128第一章研究背景及目的................................................................................129第二章研究動機............................................................................................1312-1聚亞胺（Polyimine/PolySchiffbase）..................................................1312-2Polyethyleneimine（PEI）.........................................................................1322-3Polyaminoimine（PAI）之設計..............................................................133第三章實驗流程............................................................................................1343-1實驗藥品................................................................................................1353-2實驗儀器與裝置....................................................................................1353-3名詞對照................................................................................................1373-4實驗步驟................................................................................................1373-4-1Benzoicaldehyde-PEI之合成...........................................................1373-4-1-1Benzoicaldehyde-PEI423之合成.................................................1373-4-1-2Benzoicaldehyde-PEI600之合成.................................................1383-4-2PAI之合成.........................................................................................1393-4-2-1PAI-1之合成...............................................................................1393-4-2-2PAI-2之合成...............................................................................1393-4-2-3PAI-3之合成..............................................................................1403-4-3材料合成之鑑定..............................................................................1413-4-4PAI之水解測試................................................................................1413-4-5PlasmidDNA之製備........................................................................1413-4-6PAI/DNA複合體之製備..................................................................1423-4-7Gelelectrophoresis分析....................................................................1423-4-8PAI水解後Gelelectrophoresis分析.................................................1423-4-9粒徑分析............................................................................................1433-4-10PAI之酸鹼滴定（緩衝能力）分析...................................................1433-4-11細胞培養........................................................................................1433-4-12細胞毒性測試（XTTassay）...........................................................1443-4-13PAI水解後之細胞毒性測試（XTTassay）………………….........1443-4-14PAI/DNA複合體之體外基因轉染試驗........................................1443-4-15PEI25k/PAI-1/DNA複合體之製備................................................1453-4-16PEI25k/PAI-1/DNA複合體之粒徑分析........................................1453-4-17PEI25k/PAI-1/DNA複合體之細胞毒性分析................................1453-4-18PEI25k/PAI-1/DNA複合體之體外基因轉染試驗........................1463-4-19Method1與Method2之競爭電泳測試...........................................146第四章結果與討論........................................................................................1474-1Benzoicaldehyde-PEI之合成.................................................................1474-2PAI之合成...............................................................................................1494-3PAI之水解測試.......................................................................................1574-4PAI/DNA複合體之製備........................................................................1584-4-1Gelelectrophoresis分析....................................................................1584-4-2水解之Gelelectrophoresis分析......................................................1614-4-3粒徑分析..........................................................................................1634-5PAI之酸鹼滴定（緩衝能力）分析...........................................................1644-6細胞毒性測試（XTTassay）....................................................................1654-7PAI/DNA複合體之體外基因轉染試驗................................................1674-8PEI25k/PAI-1/DNA複合體之體外基因轉染試驗.................................1704-9PEI25k/PAI-1/DNA複合體之粒徑測試.................................................1704-10PEI25k/PAI-1/DNA複合體之細胞毒性分析…………………..........1714-11PEI25k/PAI-1/DNA複合體之體外基因轉染測試...............................1724-12Method1與Method2之競爭電泳測試...............................................173第五章結論....................................................................................................177第六部份利用超音波基因傳遞技術改良基因傳遞系統之研究...............................................................................................179第一章研究背景與目的................................................................................1801-1超音波療法............................................................................................1811-1-1空穴效應..........................................................................................1811-1-2壓電材料..........................................................................................1831-2陽離子型高分子基因傳遞載體............................................................185第二章研究動機............................................................................................187第三章實驗流程............................................................................................1883-1實驗藥品................................................................................................1893-2實驗儀器與裝置....................................................................................1893-3名詞對照................................................................................................1903-4實驗步驟................................................................................................1903-4-1陽離子型高分子（PEI、PDMAEMA）/DNA複合體之製備...........1903-4-2PZT電壓操作....................................................................................1913-4-3陽離子型高分子/DNA複合穩定性試驗.......................................1913-4-3-1膠體電泳分析............................................................................1923-4-3-2粒徑測定分析............................................................................1923-4-4細胞存活率測試.............................................................................1923-4-5體外DNA傳遞實驗........................................................................1933-4-6功率、電壓、阻抗之相互關係計算..................................................1933-4-7不同電壓超音波之細胞毒性分析..................................................1943-4-8高分子/DNA複合體在不同電壓超音波下之轉染測試………...1943-4-9不同波形之超音波對高分子/DNA複合體之細胞毒性分析…...1943-4-10不同波形之超音波對高分子/DNA複合體之轉染影響測試…...195第四章結果與討論........................................................................................1964-1超音波對陽離子型高分子/DNA複合體穩定性試驗.........................1964-2超音波對細胞之毒性分析....................................................................1984-3高分子/DNA複合體在超音波下之轉染測試.......................................2004-4總功率、電壓、阻抗之相互關係計算....................................................2024-5不同電壓超音波之細胞毒性分析........................................................2034-6高分子/DNA複合體在不同電壓超音波下之轉染測試.....................2034-7不同波形之超音波對高分子/DNA複合體之細胞毒性分析.............2054-8不同波形之超音波對高分子/DNA複合體之轉染影響測試…….....206第五章結論....................................................................................................209第七部份新穎奈米型藥物釋控材料PEG-dsDNA/Lipofectamine之開發及細胞內藥物釋控之研究....................................................................211第一章研究背景與目的................................................................................2121-1奈米化藥物............................................................................................2121-1-1微酯粒（liposome）............................................................................2171-1-2奈米微胞（micelles）.........................................................................2201-1-3奈米碳管/碳球（nanotube）及其他奈米材料...................................2211-2藥物釋控................................................................................................2231-2-1利用擴散機制(Diffusioncontrolled)............................................2251-2-2利用逆滲透壓原理(Osmoticallycontrolled).................................2251-2-3利用化學方式(Chemicallycontrolled)..........................................2251-2-3-1化學鍵斷腐蝕方式....................................................................2261-2-3-2離子交換樹脂..............................................................................2261-2-4藥物載體..........................................................................................2261-2-4-1天然高分子..................................................................................2241-2-4-2合成膠..........................................................................................2241-2-4-3高分子合成..................................................................................2241-2-5可分解性交聯劑之載體..................................................................2271-2-6可分解性骨架（backbone）之載體...................................................2271-2-7可分解性支鏈載體..........................................................................228第二章研究動機............................................................................................2292-1PEG介紹..................................................................................................229第三章實驗流程............................................................................................2343-1實驗藥品................................................................................................2353-2實驗儀器與裝置....................................................................................2363-3名詞對照................................................................................................2373-4實驗步驟................................................................................................2373-4-1PEG-diNCO之合成..........................................................................2373-4-1-1PEG4k-diNCO之合成....................................................................2373-4-1-2PEG10k-diNCO之合成.................................................................2383-4-1-3PEG35k-diNCO之合成.................................................................2383-4-2PEG-dimaleimide之合成..................................................................2393-4-2-1PEG4k-dimaleimide之合成.........................................................2393-4-2-2PEG10k-dimaleimide之合成........................................................2403-4-2-3PEG35k-dimaleimide之合成........................................................2403-4-3PEG-ssDNA之合成..........................................................................2413-4-3-1ssDNA之製備.............................................................................2413-4-3-2PEG4k-ssDNA之合成..................................................................2413-4-3-3PEG10k-ssDNA之合成................................................................2413-4-3-4PEG35k-ssDNA之合成................................................................2423-4-4材料合成之鑑定..............................................................................2423-4-5PEG-6FAM-dsDNA之製備..............................................................2423-4-6EtBr染色鑑定....................................................................................2433-4-7ssDNA的裂解測試............................................................................2433-4-8細胞外藥物釋控測試......................................................................2433-4-9PEG-6FAM-dSDNA/lipofectaminecomplex之製備及粒徑測試..2443-4-10PEG-6FAM-dsDNA/lipofectamine之細胞外釋控試驗................2443-4-11細胞培養........................................................................................2443-4-12細胞毒性測試（XTTassay）...........................................................2453-4-13細胞內藥物釋控分析....................................................................2353-4-14細胞內PEG-6FAM-dSDNA裂解電泳分析..................................246第四章結果與討論........................................................................................2474-1PEG-diNCO之合成................................................................................2474-2PEG-dimaleimide之合成........................................................................2504-3PEG-ssDNA之合成................................................................................2544-4EtBr染色鑑定.........................................................................................2554-5ssDNA的裂解測試.................................................................................2574-6細胞外藥物釋控測試............................................................................2584-7PEG-6FAM-dSDNA/lipofectaminecomplex之製備及粒徑測試…....2614-8PEG-6FAM-dsDNA/lipofectamine之細胞外釋控試驗........................2634-9細胞毒性測試（XTTassay）...................................................................2634-10細胞內藥物釋控分析..........................................................................2644-11細胞內PEG-6FAM-dSDNA裂解電泳分析........................................268第五章結論....................................................................................................270第八部份附錄................................................................................................2711.質體DNA之製備.......................................................................................2711-1菌株保存與保養....................................................................................2711-2宿主細胞E.coli（DH5）α之製備..........................................................2711-3pCMV-LacZ之大量製備........................................................................2721-4.質體DNA之濃度標定..........................................................................2741-5plasmidDNA的完整序列......................................................................2752.細胞株.........................................................................................................2762-1細胞種類................................................................................................2762-2細胞培養................................................................................................2772-2-1細胞培養液之配製..........................................................................2772-2-2解凍細胞..........................................................................................2772-2-3繼代培養..........................................................................................2783.DNA膠體電泳（Agarosegelelectrophoresisassays）………….................2793-1原理........................................................................................................2793-2高分子與DNA形成複合體能力的探討...............................................2803-3分析條件..................................................................................................2814.粒徑分析.....................................................................................................2814-1原理........................................................................................................2814-2分析方法................................................................................................2825.陽離子型高分子對哺乳類動物細胞毒性評估（XTTassay）…………....2835-1原理........................................................................................................2835-2分析方法................................................................................................2846.ONPG染色原理...........................................................................................2856-1分析方法..................................................................................................286第九部分參考文獻........................................................................................288圖表目錄第一部分緒論圖1、基因治療概念圖..........................................................................................6圖2、目前常見的基因傳遞方式..........................................................................7圖3、LentiviralVector的基因傳遞意示圖......................................................10圖4、一般常見的陽離子行高分子基因載體....................................................15圖5.polycation/DNA複合物轉染機制圖.......................................................16圖6.The“ProtonSponge”effect.......................................................................17第二部份陽離子型聚氨基甲酸酯之合成及其結構上胺基對DNA轉染之影響圖1、不同側鏈之PU基因載體合成.................................................................24圖2、不同側鏈之PU-glycidol基因載體合成...................................................25圖3、polyurethane（PU主鏈之合成圖）.............................................................37圖4、PU之FT-IR圖...........................................................................................38圖5、PU之1H-NMR圖表..................................................................................38圖6、三種不同側鏈之PU..................................................................................39圖7、PU1、PU2、PU3之FT-IR圖.......................................................................40圖8、PU1之1H-NMR圖....................................................................................41圖9、PU2之1H-NMR圖....................................................................................42圖10、PU3之1H-NMR圖..................................................................................42圖11、PUg1、PUg2、PUg3之合成......................................................................43圖12、PUg1、PUg2、PUg3之FT-IR圖...............................................................44圖13、PUg1之1H-NMR圖................................................................................45圖14、PUg2之1H-NMR圖................................................................................46圖15、Pug3之1H-NMR圖.................................................................................46圖16、Agarosegelelectrophoresisofpolymer/DNAcomplex..........................48圖17、高分子對DNA保護電泳測試圖(A)andDNaseI(B)protectionassayofpolymer/DNAcomplexes.................................................................50圖18、高分子/DNA複合體之粒徑分析圖......................................................51圖19、高分子酸-鹼滴定圖表............................................................................52圖20、高分子水解電泳圖..................................................................................54圖21、高分子之細胞毒性圖..............................................................................56圖22、高分子體外基因傳遞圖..........................................................................57第三部份利用Polyethylenimine與陽離子型聚胺基甲酸酯之掺合技術來增加DNA傳遞效率及降低細胞毒性之研究圖1、PUg1及PUg3之轉染效率圖......................................................................64圖2、polyurethane（PU主鏈之合成圖）.............................................................73圖3、PUg3之合成圖...........................................................................................74圖4、method1,method2andmethod3的膠體電泳分析.................................75圖5、不同掺合技術所形成的複合體類型........................................................76圖6、高分子/DNA複合體之粒徑分析圖........................................................77圖7、method1、method3以及PEI25k之細胞毒性分析圖................................78圖8、method1、method3、PEI25k的轉染分析圖................................................79第四部份新穎陽離子型聚胺基甲酸酯接枝PEI之合成與複合式基因傳遞系統之開發圖1、直鏈型與分支行PEI之合成.....................................................................84圖2、Knorr等人利用乙縮醛-PEG的試劑與PEI結合.....................................85圖3、利用雙硫鍵修飾的(A)分枝型以及(B)直鏈型PEI.................................85圖4、PU-PEI600之合成設計圖...........................................................................88圖5、PU-PEI600於複合式基因傳輸載体之結構設計圖....................................89圖6、polyurethane（PU主鏈之合成圖）............................................................102圖7、PU-PEI600之合成圖..................................................................................103圖8、PU-PEI600之FT-IR圖................................................................................103圖9、PU-PEI600之1H-NMR圖...........................................................................104圖10、PU-PEI600/DNA複合體之電泳分析圖.................................................105圖11、PU-PEI600/DNA之粒徑分析圖............................................................106圖12、PU-PEI600之酸鹼滴定（緩衝能力）分析...............................................107圖13、PU-PEI600/DNA複合體之細胞毒性圖................................................108圖14、PU-PEI600/DNA複合體之體外基因轉染試驗....................................109圖15、經由冷凍乾燥後的PU-PEI600及其轉染效率.......................................111圖16、PU-PEI600-maleimide之合成.................................................................111圖17、PU-PEI600-maleimide之FT-IR圖..........................................................112圖18、PU-PEI600-maleimide之1H-NMR圖.....................................................113圖19、PU-PEI600-maleimide/DNA複合體之電泳圖......................................114圖20、PU-PEI600-maleimide/DNA複合體之粒徑分析圖..............................115圖21、PU-PEI600-maleimide/DNA複合體之體外基因轉染試驗..................116圖22、複合式基因複合體之粒徑分析圖........................................................117圖23、複合式基因複合體之細胞毒性圖........................................................118圖24、diSH化合物之細胞毒性圖...................................................................119圖25、複合式DNA體外基因轉染試驗（pCMV-βgal）...................................120圖26、複合式基因傳遞之螢光顯微鏡圖........................................................122圖27、複合式基因傳遞之螢光顯微鏡圖........................................................125第五部份合成與鑑定一系列新型生物可降解性聚胺基亞胺之基因載體圖1、亞胺；施夫鹼（imine/Schiffbase）之結構...............................................131圖2、imine水解機制........................................................................................132圖3、新穎Polyaminoimine（PAI）基因載體之設計......................................133圖4、Benzoicaldehyde-PEI之反應流程.........................................................147圖5、Benzoicaldehyde-PEI之FT-IR圖...........................................................148圖6、Benzoicaldehyde-PEI之1H-NMR圖......................................................149圖7、PAI之反應流程.......................................................................................150圖8、PAI之FT-IR圖........................................................................................152圖9、PAI之1H-NMR圖...................................................................................153圖10、PAI-1於室溫反應之1H-NMR圖..........................................................154圖11、膠體過濾法原理圖................................................................................155圖12、PAI系列與PEI25k之GFC分析圖.......................................................156圖13、PAI之水解速率圖.................................................................................158圖14、PAI/DNA複合體之電泳圖..................................................................160圖15、水解之Gelelectrophoresis分析............................................................162圖16、PAI/DNA複合體之粒徑分析圖..........................................................164圖17、PAI之緩衝能力圖.................................................................................165圖18、PAI的細胞毒性分布圖.........................................................................166圖19、PAI水解後之細胞毒性分布圖.............................................................167圖20、PAI系列/DNA複合體之轉染效率圖..................................................169圖21、不同掺合技術所形成的複合體類型....................................................170圖22、PEI25k/PAI/DNA複合體之粒徑分析圖..............................................171圖23、PEI25k/PAI/DNA複合體之細胞毒性圖..............................................172圖24、PEI25k/PAI-1/DNA複合體之體外基因轉染測試...............................173圖25、Method1與Method2之競爭電泳測試.............................................175第六部份利用超音波基因傳遞技術改良基因傳遞系統之研究圖1、超音波之基因傳遞圖..............................................................................183圖2、A：正壓電效應；B：逆壓電效應...............................................................184圖3、壓電材料之機械能與電能之關係..........................................................185圖4、市售的PZT材料.......................................................................................185圖5、三種常見的陽離子型高分子基因載體..................................................186圖6、利用PZT產生超音波來幫助體外基因轉染的效率.............................191圖7、超音波對陽離子型高分子/DNA複合體穩定性試驗..........................197圖8、PZT運用於細胞實驗之裝置圖..............................................................199圖9、在12V電壓下三種PZT對細胞毒性之影響..........................................200圖10、C-PZT對高分子/DNA轉染效率圖.....................................................201圖11、改變電壓後的細胞毒性圖....................................................................203圖12、改變電壓後的細胞轉染圖....................................................................204圖13、兩種不同波形及其面積........................................................................205圖14、相同電壓下，不同波形對細胞的毒性測試圖......................................206圖15、相同電壓下，不同波形的轉染效率比較圖..........................................208圖16、不同電壓、相同波頂高度之轉染效率比較..........................................208第七部份新穎奈米型藥物釋控材料PEG-dsDNA/Lipofectamine之開發及細胞內藥物釋控之研究圖1、三種不同胞飲路徑圖..............................................................................215圖2、2不同特性的奈米顆粒進入細胞方式...................................................216圖3、奈米化藥物商品作用方式：左為奈米藥物，右為奈米化妝品..............216圖4、左為lipid單體構造，右為liposome完整構造........................................217圖5、liposome與細胞膜作用的方式..............................................................219圖6、高分子微胞包覆疏水性藥物的型態......................................................221圖7、三種不同的結構奈米碳管......................................................................222圖8、一般金奈米在穿透式電子顯微鏡（TEM）下型態..................................222圖9、利用MRI檢測SPIO在老鼠體內的影像圖..............................................223圖10、一般藥物與釋控型藥物作用分布圖....................................................224圖11、左:擴散機制，右:逆滲透壓原理........................................................225圖12、化學鍵斷裂方式或腐蝕方式................................................................226圖13、可分解性交聯劑之載體（上）與可分解性骨架之載體（下）......................................................................................................228圖14、可分解性支鏈載體................................................................................228圖15、DNA鹼基配對圖..................................................................................230圖16、左：膨潤型DNA水膠，右：收縮型DNA水膠......................................232圖17、PEG-dsDNA藥物搭載的平台..............................................................232圖18、PEG-dsDNA奈米化方式......................................................................233圖19、PEG-diNCO之合成................................................................................247圖20、PEG-diNCO之FT-IR圖..........................................................................248圖21、PEG-diNCO之1H-NMR圖.....................................................................249圖22、PEG-dimaleimide之合成.......................................................................250圖23、PEG-dimaleimide之FT-IR......................................................................252圖24、PEG-dimaleimide之1H-NMR.................................................................253圖25、PEG-ssDNA之合成...............................................................................254圖26、PEG-dimaleimide與PEG-ssDNA之UV圖.............................................255圖27、PEG-6FAM-dSDNA之製備方法..........................................................254圖28、2.8％agarosegel電泳分析.................................................................256圖29、ssDNA的裂解測試電泳圖...................................................................257圖30、PEG-6FAM-dsDNA釋控原理..............................................................259圖31、PEG-6FAM-dsDMA細胞外釋控圖......................................................260圖32、三種不同主鏈PEG-dsDNA的螢光釋控圖.........................................261圖33、DNase處於3天後之活性電泳圖.........................................................261圖34、PEG-6FAM-dsDNA/lipofectamine的粒徑分析圖..............................262圖35、PEG-6FAM-dsDNA/lipofectamine之細胞外釋控試驗......................263圖36、PEG10k-6FAM-dsDNA/lipofectamine的細胞毒性分析圖..................264圖37、PEG10k-6FAM-dsDNA在細胞內釋控情形..........................................265圖38、PEG10k-6FAM-dsDNA在細胞內釋控的速率圖..................................267圖39、liposome將DNA送入細胞之機制.......................................................267圖40、細胞內PEG-6FAM-dSDNA裂解電泳分析.........................................269第八部份附錄圖1、pCMV-β-gel的完整序列.........................................................................275圖2、pEGFP-C2的完整序列...........................................................................275圖3、pLanRFP-N的完整序列..........................................................................276圖4、COS-7細胞生長情形............................................................................277圖5、螢光化合物ethidiumbromide的化學結構式.....................................280圖6、XTT的化學結構式.................................................................................283圖7、XTT在細胞中反應形成formazan鹽類.................................................284圖8、ONPG對半乳糖分解酵素作用之原理..................................................286表1、LuriaBroth的組成.................................................................................271表2、QIAGENplasmidmegakit......................................................................273表3、XTT試劑的組成.....................................................................................285 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電子全文國圖紙本論文推文網路書籤推薦評分引用網址轉寄 top 相關論文相關期刊熱門點閱論文 1. 超音波穴蝕效應於基因傳遞效率之研究 2. 超音波微氣泡之製備與生醫應用 3. 生物可分解性陽離子型高分子載體之合成及應用在基因傳遞之研究 4. 純鎂製骨釘/骨板於模擬體液之間隙腐蝕及其生成物對骨釘與骨板間抗拉強度影響之研究 5. PEI-PLGA奈米微粒:以不同分子量的PEI製成之奈米微粒特性與細胞吸收 6. 無機離子添加於陽離子性PLGA奈米微粒做為基因傳輸材料之研究 7. 含胺基甲酸酯及短鏈PEI之生物可分解陽離子型聚酯合成及其DNA轉染能力之研究 8. 陽離子性PLGA奈米微粒之製備及做為基因傳輸材料之研究 9. Conjugationofviralpeptidetoimprovethepolymeric-mediatedgenedelivery 10. 陽離子型聚酯材料合成、改質及基因傳輸之研究 11. 新生物可分解性陽離子型聚胺基甲酸酯高分子載體合成與基因輸送應用之研究 1. 28. 李福鐘，「”五反”、群眾動員與中共高層政治」，輔仁歷史學報（新北），第15期，2004年7月，頁239-290。

2. 蔣義斌，＜天臺宗懺儀與身體＞，《佛學研究中心學報》第十三期，台北：鴻霖出版，2007年。

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