現在位置首頁 > 博碩士論文 > 詳目
  • 同意授權
論文中文名稱:經電擊處理後之微脂體對轉染效率的影響之研究 [以論文名稱查詢館藏系統]
論文英文名稱:The effects of electric-treated DNA-liposome complex on transfection efficiency [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:100
出版年度:101
中文姓名:黃采瑜
英文姓名:Tsai-Yu Huang
研究生學號:99688002
學位類別:碩士
語文別:中文
口試日期:2012-07-27
論文頁數:94
指導教授中文名:侯劭毅
口試委員中文名:黃志宏;王勝仕;黃光策
中文關鍵詞:pIRES2-EGFPLipofectamine 2000粒徑大小轉染效率
英文關鍵詞:pIRES2-EGFPLipofectamine 2000particle sizetransfection efficiency
論文中文摘要:目前已有許多動物細胞的轉染技術,包括利用病毒性載體(例如:腺病毒或反轉錄病毒等)、物理性基因傳遞的方法(例如:電擊、基因槍或微泡等)或使用商業上常用的化學轉染試劑(例如:聚合物或陽離子型的微脂體等)。而在轉染的過程中有許多影響轉染效率的因子,若以微脂體為例,以其粒徑大小和轉染後的培養時間等最被廣為研究。
在此,我們以在哺乳動物細胞能表現綠色螢光蛋白的pIRES2-EGFP作為表現質體,並使用Lipofectamine 2000作為攜帶此質體的轉染試劑。本實驗研究中,我們證明了電擊後的DNA-liposome complex粒徑大小會增加;不過,轉染效率反而下降。從許多結果顯示出不同於發表的文獻所提出的DNA-liposome complex粒徑大小增加會提高轉染效率的論點。
論文英文摘要:There are many transfection technologies of animal cell, including the use of viral vectors, such as adenovirus and retrovirus, physical gene delivery method, such as electroporation, gene gun and microbubbles, and commercial chemical transfection reagents, such as polymer and cationic liposome. Many factors affect transfection efficiency in the transfection process. For example, the size of liposome particles and incubation time after transfection are most widely studied.
Here we used pIRES2-EGFP as an expression plasmid that could produce green fluorescent protein in mammalian cells, and Lipofectamine 2000 as the transfection reagents. In this study, we demonstrate that the size of DNA-liposome complex increased after electric shock; however, the transfection efficiency decreased. These results are different from the researchs which reported that the size of DNA-liposome complex increased, the transfection efficiency increased.
論文目次:目錄
摘要.....................................................i
ABSTRACT.................................................ii
誌謝.....................................................iii
目錄.....................................................iv
表目錄...................................................vii
圖目錄....................................................x
第一章 緒論..............................................1
1.1 前言..................................................1
1.2 研究動機及目的........................................2
第二章 研究背景與原理介紹................................3
2.1 細胞轉染系統分類......................................3
2.2 微脂體介紹............................................7
2.2.1 陽離子型微脂體.............................7
2.2.2 陽離子型微脂體-DNA複合物的形成.............7
2.2.3 陽離子型微脂體的轉染機制...................7
2.2.4 影響轉染效率的因子.........................9
2.2.5 陽離子型微脂體-DNA複合物電擊處理的轉染機制.10
2.3 細胞形態與轉染效率分析................................12
2.3.1 光學顯微鏡.................................12
2.3.2 螢光顯微鏡.................................12
2.4 細胞毒性分析..........................................15
2.4.1 細胞存活率(MTT assay)......................15
2.4.2 細胞死亡率(LDH assay)......................16
2.5 粒徑大小檢測原理......................................17
第三章 實驗方法與步驟....................................18
3.1 實驗架構..............................................18
3.1.1 質體pIRES2-EGFP純化.........................18
3.1.2 L929細胞培養...............................20
3.1.2.1 冷凍細胞活化.........................20
3.1.2.2 細胞繼代.............................21
3.1.2.3 冷凍細胞.............................21
3.1.2.4 細胞計數.............................22
3.1.3 轉染方法...................................23
3.1.3.1 轉染前一天...........................24
3.1.3.2 轉染當天.............................25
3.1.3.2.1 DNA-lipofectamineTM 2000
complexes的製備...........25
3.1.3.2.2 轉染......................27
3.1.4 螢光顯微鏡觀察細胞形態與轉染效率分析.......28
3.1.5 細胞毒性分析...............................28
3.1.5.1 細胞存活率(MTT assay)................28
3.1.5.2 細胞死亡率(LDH assay)................30
3.1.6 粒徑大小檢測...............................32
3.2 實驗藥品與設備........................................33
3.2.1 藥品.......................................33
3.2.2 設備.......................................35
第四章 結果與討論........................................37
4.1實驗過程中遭遇的問題...................................37
4.2細胞形態與轉染效率分析.................................38
4.2.1 固定DNA用量並調整LF2000用量................39
4.2.1.1 Lipoplexes未藉電擊處理後的轉染效率...39
4.2.1.2 Lipoplexes藉電擊處理後的轉染效率.....40
4.2.2 固定LF2000用量並調整DNA用量................42
4.2.2.1 Lipoplexes未藉電擊處理後的轉染效率...43
4.2.2.2 Lipoplexes藉電擊處理後的轉染效率.....44
4.3 細胞毒性分析..........................................47
4.3.1 細胞存活率(MTT assay)......................47
4.3.1.1固定DNA用量並調整LF2000用量...........47
4.3.1.1.1 Lipoplexes未藉電擊處理後的細胞
存活率......................47
4.3.1.1.2 Lipoplexes藉電擊處理後的細胞存
活率........................49
4.3.1.2 固定LF2000用量並調整DNA用量..........51
4.3.1.2.1 Lipoplexes未藉電擊處理後的細胞
存活率......................51
4.3.1.2.2 Lipoplexes藉電擊處理後的細胞存
活率........................53
4.3.2 細胞死亡率(LDH assay)......................56
4.3.2.1 固定DNA用量並調整LF2000用量..........56
4.3.2.1.1 Lipoplexes未藉電擊處理後的細胞
死亡率......................56
4.3.2.1.2 Lipoplexes藉電擊處理後的細胞死
亡率........................58
4.3.2.2 固定LF2000用量並調整DNA用量..........60
4.3.2.2.1 Lipoplexes未藉電擊處理後的細胞
死亡率......................60
4.3.2.2.2 Lipoplexes藉電擊處理後的細胞死
亡率........................62
4.4 粒徑大小檢測..........................................65
4.4.1 固定DNA用量並調整LF2000用量................65
4.4.1.1 Lipoplexes電擊處理前後的粒徑大小.....65
4.4.2 固定LF2000用量並調整DNA用量................67
4.4.2.1 Lipoplexes電擊處理前後的粒徑大小.....67
第五章 結論...............................................70
參考文獻..................................................72
附錄......................................................84
附錄一 固定DNA用量並調整LF2000比例;Lipoplexes未藉電擊處理,於轉染後48小時的顯微鏡影像(100X)....................85
附錄二 固定DNA用量並調整LF2000比例;Lipoplexes藉電擊(450V; 15μF)處理後,於轉染後48小時的顯微鏡影像(100X)..........88
附錄三 固定LF2000用量並調整DNA比例;Lipoplexes未藉電擊處理,於轉染後48小時的顯微鏡影像(100X)....................90
附錄四 固定LF2000用量並調整DNA比例;Lipoplexes藉電擊(450V; 15μF)處理後,於轉染後48小時的顯微鏡影像(100X)。........93

表目錄
表2-1 細胞轉染方法........................................4
表3-1 Gene-Spin™ Miniprep Purification Kit–V2內容物.....19
表3-2 此研究的轉染條件設定...............................25
表3-3 In Vitro Toxicology Assay Kit, Lactic Dehydrogenase based內容物...............................................30
表3-4 實驗中所使用的材料與試劑...........................33
表3-5 實驗中所使用的儀器與設備...........................35
表4-1 固定DNA用量並調整LF2000比例;Lipoplexes未藉電擊處理後的轉染效率................................................40
表4-2 固定DNA用量並調整LF2000比例;Lipoplexes藉電擊(450V;15μF)處理後的轉染效率...................................41
表4-3 固定LF2000用量並調整DNA比例;Lipoplexes未藉電擊處理後的轉染效率................................................44
表4-4 固定LF2000用量並調整DNA比例;Lipoplexes藉電擊(450V;15μF)處理後的轉染效率...................................45
表4-5 固定DNA用量並調整LF2000比例;Lipoplexes未藉電擊處理後的細胞存活率(轉染後約24小時)............................48
表4-6 固定DNA用量並調整LF2000比例;Lipoplexes未藉電擊處理後的細胞存活率(轉染後約48小時)............................48
表4-7 固定DNA用量並調整LF2000比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞存活率(轉染後約24小時)...............49
表4-8 固定DNA用量並調整LF2000比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞存活率(轉染後約48小時)...............50
表4-9 固定LF2000用量並調整DNA比例;Lipoplexes未藉電擊處理後的細胞存活率(轉染後約24小時..............................52
表4-10 固定LF2000用量並調整DNA比例;Lipoplexes未藉電擊處理後的細胞存活率(轉染後約48小時)............................53
表4-11 固定LF2000用量並調整DNA比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞存活率(轉染後約24小時)...............54
表4-12 固定LF2000用量並調整DNA比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞存活率(轉染後約48小時)...............54
表4-13 固定DNA用量並調整LF2000比例;Lipoplexes未藉電擊處理後的細胞死亡率(轉染後約24小時)............................57
表4-14固定DNA用量並調整LF2000比例;Lipoplexes未藉電擊處理後的細胞死亡率(轉染後約48小時)............................57
表4-15 固定DNA用量並調整LF2000比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞死亡率(轉染後約24小時)...............58
表4-16 固定DNA用量並調整LF2000比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞死亡率(轉染後約48小時)...............59
表4-17 固定LF2000用量並調整DNA比例;Lipoplexes未藉電擊處理後的細胞死亡率(轉染後約24小時)............................61
表4-18 固定LF2000用量並調整DNA比例;Lipoplexes未藉電擊處理後的細胞死亡率(轉染後約48小時..............................62
表4-19 固定LF2000用量並調整DNA比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞死亡率(轉染後約24小時)...............63
表4-20 固定LF2000用量並調整DNA比例;Lipoplexes藉電擊(450V;15μF)處理後的細胞死亡率(轉染後約48小時)...............63
表4-21 固定DNA用量並調整LF2000比例;Lipoplexes電擊處理前後的粒徑大小..................................................66
表4-22 固定LF2000用量並調整DNA比例;Lipoplexes電擊處理前後的粒徑大小..................................................68

圖目錄
圖2-1 Lipoplex調節的細胞轉染與內吞作用....................9
圖2-2 質體pIRES2-EGFP map................................11
圖2-3 Lipoplexes電擊處理轉染步驟概要圖...................11
圖2-4 螢光顯微鏡原理.....................................13
圖2-5 螢光發光能階示意圖.................................14
圖2-6 MTT化學結構與其還原態..............................15
圖2-7 LDH分析反應概要圖.................................16
圖2-8 粒徑大小檢測示意圖.................................17
圖3-1 生物特性分析流程圖.................................18
圖3-2 細胞轉染分析流程概述圖.............................23
圖4-1 光學顯微鏡下觀察L929 cell緻密程度之影像(1000X).....39
圖4-2 固定DNA用量並調整LF2000比例;Lipoplexes電擊處理與否的轉染效率柱狀圖:(A) 轉染後24小時;(B) 轉染後48小時........41
圖4-3 固定LF2000用量並調整DNA比例;Lipoplexes電擊處理與否的轉染效率柱狀圖:(A) 轉染後24小時;(B) 轉染後48小時........45
圖4-4 固定DNA用量並調整LF2000比例;Lipoplexes電擊處理與否的細胞存活率柱狀圖:(A) 轉染後24小時;(B) 轉染後48小時......50
圖4-5 固定LF2000用量並調整DNA比例;Lipoplexes電擊處理與否的細胞存活率柱狀圖:(A) 轉染後24小時;(B) 轉染後48小時......55
圖4-6 固定DNA用量並調整LF2000比例;Lipoplexes電擊處理與否的細胞死亡率柱狀圖:(A) 轉染後24小時;(B) 轉染後48小時......59
圖4-7 固定LF2000用量並調整DNA比例;Lipoplexes電擊處理與否的細胞死亡率柱狀圖:(A) 轉染後24小時;(B) 轉染後48小時......64
圖4-8 固定DNA用量並調整LF2000比例;Lipoplexes電擊處理前後的粒徑大小曲線趨勢圖........................................67
圖4-9 固定LF2000用量並調整DNA比例;Lipoplexes電擊處理前後的粒徑大小曲線趨勢圖........................................69
論文參考文獻:[1] A.D. Miller, 2003. The problem with cationic liposome/micelle-based non-viral vector systems for gene therapy. Curr. Med. Chem. 10: 1195-1211.
[2] Alan L. Parker, Christopher Newman, Simon Briggs, Leonard Seymour and Paul J. Sheridan, 2003. Lipoplex-mediated transfection and endocytosis. Molecular Medicine 5
[3] Almofti MR, Harashima H, Shinohara Y, Almofti A, Li W, Kiwada H, 2003. Lipoplex size determines lipofection efficiency with or without serum. Mol Membr Biol. 20: 35–43.
[4] Almofti MR, Harashima H, Shinohara Y, Almofti A, Baba Y, Kiwada H,
2003. Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization. Arch Biochem Biophys. 410: 246–253.
[5] A. Masotti, G. Mossa, C. Cametti, G. Ortaggi, A. Bianco, N. Del Grosso, D. Malizia, C. Esposito, 2009. Comparison of different commercially available cationic liposome–DNA lipoplexes: Parameters influencing toxicity and transfection efficiency. Colloids and Surfaces B: Biointerfaces 68: 136–144.
[6] Andre, N.D., Barbosa, D.S., Munhoz, E., Estevao, D., Cecchini, R., Watanabe, M.A., 2004. Measurement of cytotoxic activity in experimental cancer. J. Clin. Lab. Anal. 18: 27–30.
[7] A.V. Kabanov, P.L. Felgner, L.W. Seymour, 1998. Self-assembling Complexes for Gene delivery: From Laboratory to Clinical Trial. John Willey and Sons. New York.
[8] Bing Wang, Jiti Zhou, Shaohui Cui, Baoling Yang, Yinan Zhao, Budiao Zhao, Yan Duan and Shubiao Zhang, 2012. Cationic liposomes as carriers for gene delivery: Physico-chemical characterization and mechanism of cell transfection. African Journal of Biotechnology 11: 2763-2773.
[9] B. Martin, M. Sainlos, A. Aissaoui, N. Oudrhiri, M. Hauchecorne, J.P. Vigneron, J.M. Lehn, P. Lehn, 2005. The design of cationic lipids for gene delivery. Curr. Pharm. Des. 11: 375-394.
[10] Boktov J, Hirsch-Lerner D, Barenholz Y, 2007. Characterization of the interplay between the main factors contributing to lipoplex-mediated transfection in cell cultures. J Gene Med. 9: 884–893.
[11] Bonte F, Juliano RL, 1986. Interactions of liposomes with serum proteins. Chem Phys Lipids 40: 359–372.
[12] C.C. Christine, L. Huang, 2005. Recent advances in non-viral gene delivery. Adv. Genet. 53: 3-18.
[13] Celine Raymond and Roseanne Tom et al., 2011. A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications. Methods 55: 44–51.
[14] Chunhua Fu and Xiaoli Sun et al., 2011. Biodegradable Tri-Block Copolymer Poly(lactic acid)-poly(ethylene glycol)-poly(L-lysine)(PLA-PEG-PLL) as a Non-Viral Vector to Enhance Gene Transfection. Int. J. Mol. Sci. 12: 1371-1388.
[15] Crystal RG et al., 1994. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet 8: 42–51.
[16] Daniel A. Balazs andWT. Godbey, 2011. Liposomes for Use in Gene Delivery. Journal of Drug Delivery 2011:1-12.
[17] Decker, T., Lohmann-Matthes, M.L., 1988. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Methods 115: 61–69.
[18] D.-F. Zhi, S.-B. Zhang, B. Wang, Y.-N. Zhao, B.-L. Yang, S.-J. Yu, 2010. Transfection efficiency of cationic lipids with different hydrophobic domains in gene Delivery. Bioconjug. Chem. 21: 563-577.
[19] Dominic A. Scudiero, Robert H. Shoemaker, Kenneth D. Paul Anne Monks, Siobhan Tierney, Thomas H. Nofziger, Michael J. Currens, Donna Seniff, and Michael R. Boyd, 1988. Evaluation of a soluble tetrazolium/Formazan assay for cell growth and drug sensitivity in culture using human and other tumor lines. Cancer Research 48: 4827–4833.
[20] Douglas KL, 2008. Toward development of artificial viruses for gene therapy: a comparative evaluation of viral and non-viral transfection. Biotechnol Prog 24: 871– 883.
[21] Duzgenes N, Goldstein JA, Friend DS, Felgner PL, 1989. Fusion of liposomes containing a novel cationic lipid, N-[2,3-(Dioleyloxy) propyl]-N, N, N-trimethylammonium: induction of multivalent anions and asymmetric fusion with acidic phospholipid vesicles. Biochemistry 28: 9179–9184.
[22] E.-M. Damm, L. Pelkmans, J. Kartenbeck, A. Mezzacasa, T. Kurzchalia, A. Helenius, 2005. Clathrin- and caveolin-1eindependent endocytosis: entry of
simian virus 40 into cells devoid of caveolae. J. Cell Biol. 168: 477-488.
[23] E. Papadimitriou, S.G. Antimisiaris, J. Drug Target. 8 (2000) 335– 340.
[24] Elisabete Goncxalves, Robert J. Debs, and Timothy D. Heath, 2004. The Effect of Liposome Size on the Final Lipid/DNA Ratio of Cationic Lipoplexes. Biophysical Journal 86:1554-1563.
[25] Eliyahu H, Barenholz Y, Domb AJ, 2005. Polymers for DNA delivery. Molecules 10: 34–64.
[26] Eriko UCHIDA and Hiroyuki MIZUGUCHI et al.,.2002. Comparison of the Efficiency and Safety of Non-viral Vector-Mediated Gene Transfer into a Wide Range of Human Cells. Biol. Pharm. Bull. 25: 891—897.
[27] Farhood H, Bottega R, Epand RM, Huang L, 1992. Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity. Biochim Biophys Acta 1111: 239–246.
[28] Farhood H, Serbina N, Huang L, 1995. The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim Biophys Acta 1235: 289–295.
[29] Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringgold GM, Danielsen M, 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. 84: 7413-7417.
[30] F. Labat-Moleur, A.M. Steffan, C. Brisson, H. Perron, O. Feugeas, P. Furstenberger, F. Oberling, E. Brambilla, J.P. Behr, 1996. An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther. 3: 1010-1017.
[31] Gang Wang, Jianping Zhang, Abiche H. Dewilde, Anoop K. Pal, Dhimiter Bello, Joel M. Therrien, Susan J. Braunhut, Kenneth A. Marx, 2012. Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay. Toxicology 299: 99–111.
[32] Gascon AR, Pedraz JL, 2008. Cationic lipids as gene transfer agents: a patent review. Expert Opin Ther Pat.18: 515–524.
[33] Glasgow JN, Bauerschmitz GJ, Curiel DT, Hemminki A., 2004. Transductional and transcriptional targeting of adenovirus for clinical applications. Curr Gene Ther 4: 1–14.
[34] GREGORY GREGORIADIS, ROGHIEH SAFFIE and STEPHEN L. HART., 1996. High Yield Incorporation of Plasmid DNA within Liposomes: Effect on DNA Integrity and Transfection Efficiency. Journal of Drug Targetin 3: 469-475.
[35] Guohong Zhang and Steven R. Kain, 1996. Transfection Maximizer Increases the Efficiency of Calcium Phosphate Transfections with Mammalian Cells. BioTechniques 21: 940-945.
[36] Harutyun Melkonyan, Clemens Sorg and Martin Klempt, 1996. Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO). Nucleic Acids Research 24: 4356–4357.
[37] Heiko Mussauer, Vladimir L. Sukhorukov, and Ulrich Zimmermann, 2001. Trehalose Improves Survival of Electrotransfected Mammalian Cells. Cytometry 45:161–169.
[38] Hui SW et al., 1996. The role of helper lipids in cationicliposomemediated gene transfer. Biophys J 71: 590–599.
[39] Huth US, Schubert R, Peschka-Süss R, 2006. Investigating the uptake and intrcellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging. J. Control Release. 110: 490-504.
[40] K. Takei, V. Haucke, 2001. Clathrin-mediated endocytosis: membrane factors pull the trigger. Trends Cell Biol. 11: 385-391.
[41] I.R. Nabi, P.U. Le, 2003. Caveolae/raft-dependent endocytosis. J. Cell Biol 161: 673-677.
[42] J.A. Swanson, C. Watts, 1995. Macropinocytosis. Trends Cell Biol. 5: 424-428.
[43] J.A. Wolff, D.B. Rozema, 2007. Breaking the bonds: non-viral vectors become chemically dynamic. Mol. Ther. 16: 8-15.
[44] J.M. Dang, K.W. Leong, 2006. Natural polymers for gene delivery and tissue engineering. Adv. Drug Deliv. Rev. 58: 487-499.
[45] John Wiley & Sons, 1997. Optimization of Transfection. Current Protocols in Neuroscience A.1B.1-A.1B.3
[46] Joon Sig Choi and Eun Jung Lee et al., 2001. New Cationic Liposomes for Gene Transfer into Mammalian Cells with High Efficiency and Low Toxicity.Bioconjugate Chem .12: 108-113
[47] J.P. Behr, 1994. Gene transfer with synthetic cationic amphiphiles: prospects for gene therapy. Bioconjug. Chem. 5: 382-389.
[48] J.P. Behr, B. Demeneix, J.P. Loeffler, J. Perez-Mutul, 1989. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine coated DNA. Proc. Natl. Acad. Sci. U S A 86: 6982-6986.
[49] J.P. Richard, K. Melikov, H. Brooks, P. Prevot, B. Lebleu, L.V. Chernomordik, 2005. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280: 15300-15306.
[50] Kadriye Ciftci , Robert J. Levy, 2001. Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts. International Journal of Pharmaceutics 218: 81–92.
[51] Karmali PP, Chaudhuri A., 2007. Cationic liposomes as non-viral carriers of Gene medicines: resolved issues, open questiones, and future promises. Med Res Rev. 27: 696–722.
[52] K.D. Ee, S. Nir, D., 1993. Papahadjopoulos, Biochemistry 32: 889–899.
[53] Khalil IA, Kogure K, Akita H, Harashima H., 2006. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev. 58: 32–45.
[54] Kianoush Khosravi-Darani and Mohamaad Reza Mozafari, 2010. Calcium Based Non-viral Gene Delivery: An Overview of Methodology and Applications. Acta Medica Iranica 48: 133-141.
[55] K.K. Ewert, A. Ahmad, N.F. Bouxsein, H.M. Evans, C.R. Safinya, 2008. Non-viral gene delivery with cationic liposome-DNA complexes. Methods Mol. Biol. 433: 159-175.
[56] K. Kato, M. Nakanishi, Y. Kaneda, T. Uchida, Y. Okada, 1991. Expression of hepatitis B virus surface antigen in adult rat liver. Co-introduction of DNA and nuclear protein by a simplified liposome method. J. Biol. Chem. 266: 3361-3364.
[57] Kreppel F, Kochanek S., 2008. Modification of adenovirus gene transfer vectors with synthetic polymers: a scientific review and technical guide. Mol Ther 16: 16 –29.
[58] Lanhai Lu , Lihong Zhang, Maria Sen Mun Wai , David Tai Wai Yew, Jie Xu, 2012. Exocytosis of MTT formazan could exacerbate cell injury. Toxicology in Vitro 26: 636–644.
[59] Legendre, J.-Y. and Szoka Jr., Fe, 1995. Liposomes for gene therapy, In: Liposomes, New Systems and New Trends in their Applications. 669-692.
[60] Lewis JG et al., 1996. A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc Natl Acad Sci USA 93: 3176–3181.
[61] Li B, Li S, Tan Y, Stolz DB, Watkins SC, Block LH, et al., 2000. Lyophilization of cationic lipid-protamine-DNA (LPD) complexes. J Pharm Sci. 89: 355–64.
[62] Lin AJ, Slack NL, Ahmad A, George CX, Samuel CE, Safinya CR., 2003.
Three-dimensional imaging of lipid gene-carriers: membrane charge density controls universal transfection behavior in lamellar cationic liposome-DNA complexes. Biophys J. 84: 3307–3316.
[63] Liu F, Qi H, Huang L, Liu D., 1997. Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration.
Gene Ther. 4: 517–523.
[64] Liu, Y., Peterson, D.A., Kimura, H., Schubert, D., 1997a. Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Journal of Neurochemistry 69: 581–593.
[65] Liu, Y., Schubert, D., 1997. Cytotoxic amyloid peptides inhibit cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by enhancing MTT formazan exocytosis. Journal of Neurochemistry 69: 2285–2293.
[66] Marcel B. Bally, Yuan-Peng Zhang, Frances M. P. Wong, Spencer Kong, Ellen Wasan, Dorothy L. Reimer, 1997. Lipid/DNA complexes as an intermediate in the preparation of particles for gene transfer: an alternative to cationic liposome/DNA aggregates. Advanced Drug Delivery Reviews 24 : 275-290.
[67] Marija Brgles, Maja Šantak, Beata Halassy, Dubravko Forcic, Jelka Tomašić, 2012. Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency. International Journal of Nanomedicine 7: 393–401.
[68] Masoti A, Mossa G, Cametti C, et al., 2009. Comparison of different commercially available cationic liposome-DNA lipoplexes: parameters influencing toxicity and transfection efficiency. Colloids Surf, B. 68: 136–144.
[69] McLachlan G et al.,1995. Evaluation in vitro and in vivo of cationic liposome-expression construct complexes for cystic fibrosis gene therapy. Gene Therapy 2: 614–622.
[70] Michelle A. Hunt, Margaret J. Currie, Bridget A. Robinson, and Gabi U. Dachs, 2010. Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents. Journal of Biomolecular Techniques 21: 66–72.
[71] Mini Thomas and Qing Ge et al., 2005. Cross-linked Small Polyethylenimines: While Still Nontoxic, Deliver DNA Efficiently to Mammalian Cells in Vitro and in Vivo. Pharmaceutical Research 22: 373-380.
[72] Molinari, B.L., Tasat, D.R., Palmieri, M.A., Cabrini, R.L., 2005. Kinetics of MTTformazan exocytosis in phagocytic and non-phagocytic cells. Micron 36: 177–183.
[73] Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. Journal of
Immunological Methods 65: 55–63.
[74] M.P. Desai, V. Labhasetwar, E. Walter, R.J. Levy, G.L. Amidon, 1997. The mechanism of uptake of biodegradable microparticles in Caco-2 cells is size dependent. Pharm. Res. 14: 1568-1573.
[75] M.R. Almofti, H. Harashima, Y. Shinohara, A. Almofti, Y. Baba, H. Kiwada, 2003. Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization. Arch. Biochem. Biophys. 410: 246-253.
[76] M. Tyagi, M. Rusnati, M. Presta, M. Giacca, 2001. Internalization of HIV-1 Tat requires cell surface heparan sulfate proteoglycans. J. Biol. Chem. 276: 3254-3261.
[77] N. Duzgunes, S. Nir, Adv. Drug Deliv. Rev. 40 (1999) 3 –18.
[78] N. Higashi, J. Sunamoto, Biochim. Biophys. Acta 1243 (1995) 386– 392.
[79] Nicolau, C. and Cudd, A., 1989. Liposomes as carriers of DNA. Crit. Rev. Ther. Drug Carrier Syst. 6: 239-271.
[80] Niidome T, Huang L., 2002. Gene therapy progress and prospects: nonviral
vectors. Gene Ther. 9: 1647–1652.
[81] Nily Dan, 2002. Effect of liposome charge and PEG polymer layer thickness on cell–liposome electrostatic interactions. Biochimica et Biophysica Acta 1564: 343– 348.
[82] Patil SD, Rhodes DG, Burgess DJ., 2005. DNA-based therapeutics and DNA delivery systems: a comprehensive review. AAPS J. 7: E61–E77.
[83] PC Ross and SW Hui, 1999. Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Therapy 6: 651–659.
[84] Pecora P., 1979. Dynamic Light Scattering, Plenum Press Inc., New York.
[85] P.P. Karmali, A. Chaudhuri, 2007. Cationic liposomes as non-viral carriers of gene medicines: resolved issues, open questions, and future promises. Med. Res. Rev. 27: 696-722.
[86] Ramezani M, Khoshhamdam M, Dehshahri A, Malaekeh-Nikouei B., 2009.
The influence of size, lipid composition and bilayer fluidity of cationic
liposomes on the transfection efficiency of nanolipoplexes. Colloids Surf
B.72: 1–5.
[87] Ross PC, Hui SW., 1999. Lipoplex size is a major determinant of in vitro
lipofection efficiency. Gene Ther. 6: 651–659.
[88] Safinya CR., 2001. Structures of lipid-DNA complexes: supramolecular assembly and gene delivery. Curr Opin Struct Biol. 11: 440–448.
[89] Sandrine Audouy, Grietje Molema, Lou de Leij, Dick Hoekstra, 2000. Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability. J Gene Med 2: 465-476.
[90] S.D. Conner, S.L. Schmid, 2003. Regulated portals of entry into the cell. Nature 442: 37-44.
[91] S.J. Eastman, C. Siegel, J. Tousignant, A.E. Smith, S.H. Cheng, R.K. Scheule, 1997. Biophysical characterization of cationic lipid: DNA complexes. Biochim. Biophys. Acta 1325: 41-62.
[92] Sung-Kun Yim , Chul-Ho Yun, Taeho Ahn, Heung-Chae Jung and Jae-Gu Pan, 2005. A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide. Journal of Biochemistry and Molecular Biology 38: 366–369.
[93] Surendra Nimesh, Ramesh Chandra, 2008. Guanidinium-grafted polyethylenimine: An efficient transfecting agent for mammalian cells. European Journal of Pharmaceutics and Biopharmaceutics 68: 647–655.
[94] Susanne Roesler and Felix P. V. Koch et al., 2011. Amphiphilic, low molecular weight poly(ethyleneimine) derivatives with enhanced stability for efficient pulmonary gene delivery. J Gene Med 13: 123–133.
[95] Sushil Kumar Tripathi and R. Goyal et al., 2011. Polyglutamic acid-based nanocomposites as efficient non-viral gene carriers in vitro and in vivo. European Journal of Pharmaceutics and Biopharmaceutics 79: 473–484.
[96] S.Y. Wong, J.M. Pelet, D. Putnam, 2007. Polymer systems for gene delivery-past, present, and future. Prog. Poly. Sci. 32: 799-837.
[97] T.M. Allen, G.A. Austin, A. Chonn, L. Lin, K.C. Lee, Biochim. Biophys. Acta 1061 (1991) 56–64.
[98] T.M. Reineke, M.W. Grinstaff, 2005. Designer materials for nucleic acid delivery. Mat. Res. Soc. Bull. 30: 635-639.
[99] Tseng YC, Mozumdar S, Huang L., 2009. Lipid-based systemic delivery of siRNA. Adv Drug Deliv Rev. 61: 721–31.
[100] Van der Woude I et al., 1995. Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system. Biochim Biophys Acta 1240: 34–40.
[101] Vorotnikova, E., Rosenthal, R., Tries, M., Doctrow, S., Braunhut, S.J., 2010. Novel synthetic SOD mimetics can mitigate capillary endothelial cell apoptosis caused by ionizing radiation. Radiat. Res. 173: 748–759.
[102] Wang J, Lu Z, Wientjes MG, Au JL., 2010. Delivery of siRNA Therapeutics: Barriers and Carriers. AAPS J. 12: 492–503.
[103] Wasungu L, Hoekstra D., 2006. Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release. 116: 255–264.
[104] Wrobel I, Collins D., 1995. Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochem.Biophys.Acta. 1235: 296-304.
[105] Wu SY, McMillan NA., 2009. Lipidic systems for in vivo siRNA delivery. AAPS J. 11: 639–52.
[106] Xiao-Xiang Zhang , Thomas J. McIntosh, Mark W. Grinstaff, 2012. Functional lipids and lipoplexes for improved gene delivery. Biochimie 94: 42-58.
[107] Yang JP, Huang L., 1997. Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Therapy 4: 950–960.
[108] Yinghuan Li,Jie Wang, Yue Gao, Jiabi Zhu, M. Guillaume Wientjes,and Jessie L.-S. Au, 2011. Relationships between Liposome Properties, Cell Membrane Binding, Intracellular Processing, and Intracellular Bioavailability. The AAPS Journal 13: 585-597.
[109] 陳彥宏,不同藻類是出有機務及其萃取色素性質之差異。(2007),大仁科技大學環境管理研究所碩士論文。
[110] 廖國惠,以螢光顯微鏡研究二元磷脂質人造膜及固醇加入後的效應。(2008),國立中央大學物理研究所碩士論文。
[111] 資料來源:http://www.lin.com.tw/國祥貿易股份有限公司。
[112] 資料來源:Fluorescence Microscopy. Cambridge University, Rost.
[113] 資料來源:http://probes.invitrogen.com/handbook/sections/0001.html
論文全文使用權限:同意授權於2017-08-13起公開