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研究生： 范純聖
Fan, Chun-Sheng
論文名稱： 微型胞膜電穿孔基因轉殖晶片最佳化之研究
A Study on Optimization of In-Vitro Electroporated Microchip for Gene Transfection
指導教授： 林裕城
Lin, Yu-Cheng
學位類別： 碩士
Master
系所名稱： 工學院 - 工程科學系
Department of Engineering Science
論文出版年： 2004
畢業學年度： 92
語文別： 中文
論文頁數： 150
中文關鍵詞： 基因轉殖、奈米粒子、微機電製程技術、田口式品質工程方法
外文關鍵詞： gene delivery, microfabrication, nanoparticle
相關次數： 點閱：74  下載：5 
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    • 　　本研究開發出懸浮式與貼附式兩款微型胞膜電穿孔晶片，晶片製程採用微機電製程技術在玻璃基材上製作出薄膜電極，並利用高分子材料定義出反應槽；基因轉殖實驗上採用pEGFP-N1質體與自製的水相金奈米修飾DNA(T21)；本研究中應用田口式品質工程方法來提高轉殖率，並藉以求出各晶片之最佳轉殖的實驗參數。
    　　在懸浮式晶片實驗方面，藉由二維晶片電場穩態數值模擬與田口式轉殖實驗相互搭配下，驗證出電極尖端放電現象於胞膜電穿孔實驗，的確會增加細胞之基因轉殖效果，經田口式實驗方法求得對於小鼠纖維母細胞（NIH-3T3）的最佳轉殖參數為：1. 晶片電極為50µm與其間距，2. pEGFP-N1濃度為80µg/mL，3. 脈衝電壓為6V，4. 電脈衝數為2Pulse；經確認實驗可得平均轉殖率為40.36﹪。
    　　在貼附式晶片實驗方面，藉由三維晶片電泳電場穩態數值模擬與田口式轉殖實驗相互搭配下，驗證出每次電脈衝之前，先施以預濃縮電壓，使得晶片表面上的質體濃度增加，將可提升轉殖效果，經田口式實驗方法求得對於人類皮膚基底細胞癌細胞（BCC）的最佳轉殖參數為：1. 晶片電極為50µm與其間距，2. pEGFP-N1濃度為80µg/mL，3. 脈衝電壓為6V，4. 電脈衝數為2Pulse；經確認實驗可得平均轉殖率為35.89﹪。
    　　 實驗中，也利用上述藉由田口式實驗法求得的最佳轉殖參數進行水相金奈米修飾DNA(T21)轉殖；在懸浮式晶片實驗方面，NIH-3T3與BCC細胞株經懸浮式晶片最佳轉殖實驗參數；於紫外光-可見光（UV-Vis）光譜分析發現只有實驗組有粒徑13nm的金奈米粒子特有520nm波長吸收，於TEM電顯中，也只有在實驗組找到了金奈米的蹤跡，顯示成功的將金奈米修飾DNA(T21)轉殖進入細胞中。在貼附式晶片實驗方面，NIH-3T3與BCC細胞株經貼附式晶片最佳轉殖實驗參數；於UV-Vis光譜分析發現只有實驗組有粒徑13nm的金奈米粒子特有520nm波長吸收，且有外加電泳再行胞膜電穿孔的520nm波長吸收峰值高於純胞膜電穿孔；於TEM電顯中，也只有在實驗組找到了金奈米的蹤跡，不只顯示成功的將金奈米修飾DNA(T21)轉殖進入細胞中，更再次驗證電泳預濃縮，將可提升轉殖效果。

    　　Au nanoparticles modified with 21-base thiolated-oligonucleotides or pEGFP-N1 have been evaluated as delivery vehicles for the development of a nonviral transfection platform. The electromigration combined with electroporation for DNA delivery in BCC and NIH-3T3 cells were employed to test on microchips. Electroporation introduces foreign materials into cells by applying impulses of electric field to induce multiple transient pores on the cell membrane through dielectric breakdown of the cell membrane.
    　　On the basis of the characteristic surface plasmon of the Au particles, UV-Vis absorption was utilized to qualitatively judge the efficiency of delivery. Transmission electron microscopy images and Flow Cytometer (quantitative analysis) provided evidence of the Au/oligonucleotide nanoparticles or pEGFP-N1 before and after electroporation and electromigration function.The experiments demonstrated that electrophoretic migration followed by electroporation significantly enhanced the transportation efficiency of the　nanoparticleoligonucleotide complexes as compared with electroporation alone.

    中文摘要.............................................................I Abstract............................................................II 誌 謝..............................................................III 目 錄...............................................................IV 表目錄..............................................................IX 圖目錄..............................................................XI 第一章 緒論..........................................................1 1.1前言..............................................................1 1.2胞膜電穿孔技術....................................................4 1.2.1細胞膜特性......................................................4 1.2.2可逆性的電崩潰..................................................6 1.3研究動機與目的....................................................8 1.4研究架構.........................................................12 第二章 晶片結構設計及數值模擬.......................................13 2.1晶片結構設計.....................................................13 2.1.1懸浮式胞膜電穿孔晶片設計.......................................14 2.1.2貼附式胞膜電穿孔晶片設計.......................................16 2.2晶片電壓電場數值模擬.............................................19 2.2.1懸浮式胞膜電穿孔晶片...........................................20 2.2.1.1晶片數值模型建立.............................................20 2.2.1.2結構模型離散化...............................................21 2.2.1.3邊界條件設定與運算...........................................23 2.2.1.4模擬後處理...................................................24 2.2.2貼附式胞膜電穿孔晶片...........................................25 2.2.2.1晶片數值模型建立.............................................25 2.2.2.2結構模型離散化...............................................25 2.2.2.3邊界條件設定與運算...........................................26 2.2.2.4模擬後處理...................................................27 第三章 微晶片製作與脈衝電源系統.....................................28 3.1晶片製程.........................................................28 3.1.1晶片金屬電極製程...............................................29 3.2.2反應槽母模製作.................................................32 3.2.3模造PDMS反應槽.................................................36 3.2.4晶片封裝與滅菌.................................................41 3.3脈衝電源供應系統.................................................44 3.3.1單晶片微處理器.................................................44 3.3.2驅動電路.......................................................47 3.3.3脈衝信號產生...................................................51 第四章 質體與載體製備及其實驗分析平台建立...........................52 4.1螢光基因轉殖之質體準備...........................................52 4.2螢光偵測分析系統.................................................55 4.2.1螢光顯微鏡拍攝分析.............................................55 4.2.2流式細胞儀分析.................................................58 4.2.2.1工作原理.....................................................59 4.2.2.2分析原理.....................................................60 4.2.2.3資料分析.....................................................62 4.3金奈米修飾DNA(T21)轉殖...........................................66 4.4金奈米修飾DNA(T21)偵測分析系統...................................71 4.4.1紫外光-可見光吸收光譜分析......................................71 4.4.2穿透式電子顯微鏡拍攝分析.......................................75 第五章 細胞培養與實驗...............................................78 5.1細胞培養與收集計數...............................................78 5.1.1 繼代培養步驟..................................................79 5.1.2細胞收集與計數.................................................84 5.2懸浮式胞膜電穿孔晶片應用於基因轉殖之實驗步驟.....................87 5.3貼附式胞膜電穿孔晶片應用於基因轉殖之實驗步驟.....................89 第六章 結果與討論...................................................93 6.1細胞外觀型態觀察.................................................93 6.2晶片結構數值模擬結果分析.........................................97 6.2.1懸浮式胞膜電穿孔晶片...........................................97 6.2.2貼附式胞膜電穿孔晶片..........................................106 6.3懸浮式胞膜電穿孔晶片基因轉殖之效率評估..........................112 6.4貼附式胞膜電穿孔晶片基因轉殖之效率評估..........................121 6.5懸浮式胞膜電穿孔晶片應用於金奈米修飾硫醇核甘酸之轉殖效率評估....129 6.6貼附式胞膜電穿孔晶片應用於金奈米修飾硫醇核甘酸之轉殖效率評估....134 第七章 結論與建議..................................................141 7.1結論............................................................141 7.2建議............................................................143 參考文獻...........................................................144

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