臺灣博碩士論文加值系統

本研究探討利用電泳與胞膜電穿孔實驗，來增加基因轉殖晶片上特定區域的轉殖效果，胞膜電穿孔是一種讓細胞膜外的物質進入細胞膜內的技術，其原理利用電脈衝所形成的電場，使細胞膜表面造成可逆性的電崩潰，產生瞬間的微小孔洞來使物質進入，實驗上採用pEGFP-N1質體，在晶片上轉殖進入成骨細胞(MC3T3E)中。
本實驗利用微機電製程技術在玻璃底材上製作出薄膜電極，並利用高分子材料定義細胞反應區大小，在胞膜電穿孔前將DNA注入且蓋滿反應區，利用上下電極所產生的電場來驅動帶負電的DNA，讓DNA聚集在通正電的指叉狀晶片電極上，使DNA濃度提高，接著進行胞膜電脈衝轉殖實驗。在電泳過程中，利用一維解析解與二維數值模擬來探討DNA濃度的變化，由研究的結果顯示，在電脈衝之前，DNA在晶片表面上的濃度可增加到原本的數千倍，因此，藉由DNA電泳後濃度的提高來增加傳統電脈衝的轉殖效果，本實驗成功地利用電泳技術提高胞膜電穿孔晶片的轉殖效率。

The present paper investigated the site-specific enhancement of in vitro gene delivery using electrophoresis and electroporation microchips. Electroporation is a technique that introduces foreign materials into cells by applying impulses with an electric field to create multiple transient pores in the cell membrane through a dielectric breakdown of it. The pEGFP-N1 plasmids were transfected into osteoblasts cells (MC3T3E) on the microchip.
The electroporation chip with thin film electrodes were fabricated on a glass slide using microfabrication technologies. The region of cell culture cavity defines by rectangular macromolecular compounds. The electric field for DNA attracting results in the increment of DNA concentration on the surface of these powered electrodes. Then the electric power was switched to the interdigital electrodes to continue the electroporation process. The DNA concentration during electrophoresis on the microchip was investigated by one-dimensional steady-state approximation and two-dimensional transient simulation in this study. The study demonstrated the DNA concentration close to the cell surface increased up to several thousands-fold before electroporation so that improve the transfection efficiency comparing to the conventional electroporation. The present work successfully enhanced the site-specific transfection utilizing the attracting electric field on the electroporation microchip.

中文摘要I
AbstractII
誌謝III
目錄IV
表目錄VII
圖目錄VIII
第一章 緒論1
1.1前言 1
1.2文獻回顧3
1.3研究動機與目的5
1.4實驗架構7
第二章 理論分析 10
2.1胞膜電穿孔理論10
2.1.1細胞膜特性10
2.1.2可逆性的電崩潰12
2.2電泳理論13
2.3一維穩態解析解14
2.4二維暫態數值模擬17
2.4.1二維格點建立17
2.4.2邊界條件18
2.5結果與討論20
第三章 晶片設計與製作30
3.1晶片設計30
3.2晶片製程34
3.3晶片封裝與滅菌37
第四章 細胞培養及實驗平台建立38
4.1晶片前處理38
4.2細胞培養40
4.2.1細胞培養與收集40
4.2.2細胞計數與晶片上培養42
4.3電泳平台45
4.4 DNA與胞膜電穿孔47
4.5螢光偵測系統51
4.6流式細胞分析儀53
4.6.1流式細胞儀原理53
4.6.2流式細胞儀資料分析55
第五章 結果與討論59
5.1細胞生長59
5.2傳統轉殖實驗61
5.2.1磷酸鈣法(Calcium Phosphate)61
5.2.2微脂粒法(Liposome)63
5.3單純胞膜電穿孔65
5.4單純電泳69
5.5電泳與胞膜電穿孔78
第六章 結論與建議88
6.1結論88
6.2建議90
參考文獻91

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[37]http://www.sigmaaldrich.com
[38]http://www.biocenter.helsinki.fi/bi/peranen/Kotisivunlinkit/Vectors/pEGFP-N1.htm