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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張富雄(Fu-Hsiuan Chang) | |
| dc.contributor.author | Hsin-Hung Chen | en |
| dc.contributor.author | 陳信宏 | zh_TW |
| dc.date.accessioned | 2021-06-13T00:03:29Z | - |
| dc.date.available | 2007-08-08 | |
| dc.date.copyright | 2007-08-08 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-30 | |
| dc.identifier.citation | 1. Downward, J. (2004) Use of RNA interference libraries to investigate oncogenic signalling in mammalian cells. Oncogene, 23, 8376-8383.
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Vogel, S., Piantedosi, R., Frank, J., Lalazar, A., Rockey, D.C., Friedman, S.L. and Blaner, W.S. (2000) An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro. J Lipid Res, 41, 882-893. 58. Hsu, Y.C., Chiu, Y.T., Cheng, C.C., Wu, C.F., Lin, Y.L. and Huang, Y.T. (2007) Antifibrotic effects of tetrandrine on hepatic stellate cells and rats with liver fibrosis. J Gastroenterol Hepatol, 22, 99-111. 59. Gunther, T. (2006) Concentration, compartmentation and metabolic function of intracellular free Mg2+. Magnes Res, 19, 225-236. 60. Igamberdiev, A.U. and Kleczkowski, L.A. (2006) Equilibration of adenylates in the mitochondrial intermembrane space maintains respiration and regulates cytosolic metabolism. J Exp Bot, 57, 2133-2141. 61. Buchachenko, A.L. and Kuznetsov, D.A. (2006) [Magnesium magnetic isotope effect: a key towards mechanochemistry of phosphorylating enzymes as molecular machines]. Mol Biol (Mosk), 40, 12-19. 62. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28245 | - |
| dc.description.abstract | 基因轉殖為研究基因之功能的方法之一，能否高效率轉殖基因且不對細胞造成毒害性，是目前基因轉殖所面臨之挑戰，利用本實驗室研發之界面基因轉殖法 ( surfection )，可有效解決細胞毒性問題，而且，界面基因轉殖能有效遞送三種基因，實驗之操作程序較傳統基因轉殖技術簡單且快速。
本實驗利用正價脂質進行界面基因轉殖之分析研究，實驗操作方法係利用正價脂質塗附之界面，與正價離子與 pEGFP 綠色螢光質體基因之溶液進行混合，並利用 COS-7 細胞進行基因轉殖效率之分析，經一系列不同之正價離子實驗分析發現，濃度 100 mM 之氯化鈉、 10 mM 之氯化鎂與濃度 0.3 mM 之硫酸鋅溶液，利用 2.5×105 數量之 COS-7 細胞，於脂質界面基因轉殖時，具有較高之基因遞送效率，且不產生細胞毒性。此外，本實驗亦設立基因轉殖材料之最佳使用時間，例如：脂質塗附之界面為 2 星期、質體基因與氯化鎂混合溶液為 1 星期、混合溶液界面為 1星期，更進一步，本實驗利用牛血清白蛋白 ( BSA ) ，成功保存乾燥後界面之基因轉殖效力，完成脂質界面基因轉殖快速化之目標。而在脂質界面基因轉殖之應用上，也有諸多突破，本實驗利用脂質界面基因轉殖法，已成功對老鼠骨髓幹細胞、神經細胞與肝臟星狀細胞，進行高效率之基因轉殖。 綜合以上，本實驗成功建立脂質界面基因轉殖之標準操作程序，並依此操作程序對較難轉殖之細胞（例如：幹細胞、神經細胞與肝臟星狀細胞），進行高效率之基因遞送；另一方面，實驗批次材料之最佳使用時間、界面基因轉殖效力之保存方法，本實驗也都建立了系統性的管理，未來可望以脂質界面基因轉殖法為平檯，對細胞進行多功能性基因之遞送，以利訊息傳遞領堿之拓展研究。 | zh_TW |
| dc.description.abstract | Transfection is one way to study functions of genes. Current transfection methods are in the face of overcoming challenges to have high transfection efficiency and low cell cytotoxicity. Using surfection can solve the problem of cell cytotoxicty. Besides, surfection protocol is faster and easier than traditional transfection which can deliver three genes effectively in the meanwhile.
We took cationic lipids, DOTAP, for surfection analysis. Lipid surfection protocol is seeding COS-7 cell number 2.5×105/ well onto lipid coating surface soaking with cation and plasmid DNA mixture solution. After serial analysis by lipid surfection with different cation solutions, we found that cation solutions in specific concentration, which is 100 mM NaCl, 10 mM MgCl2 and 0.3 mM ZnSO4, will give better transfection efficiency and low cell cytotoxicity. Besides, we setup preservation deadlines of surfection materials, such as lipid coating surface, mixture solutions, and lipid coating surface with mixture solutions. Their suggested deadlines are 2 week, 1 week and 1 week, respectively. Moreover, we developed instant lipid surfection protocol. Adding 1% BSA solution onto surfection surface will preserve transfection efficacy. There are lots of breakthroughs in lipid surfection application, too. For example, high-efficiency transfection of genes to rat bone marrow stem cells ( rBMSC ) neuron cells, and hepatic stellate cells ( HSC ) is achieved by lipid surfection. In conclusion, we setup the standard operation protocols of lipid surfection ( SOP ). Next, we delivered genes to rBMSC, neuron cells and HSC in a high-efficiency manner by lipid surfection SOP. Furthermore, we established systemic management for deadlines of batch materials and preservation protocols of surfection efficacy. In the future, we may use lipid surfection as a platform to transfect multi-functional genes to cells for signal transduction advanced study. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T00:03:29Z (GMT). No. of bitstreams: 1 ntu-96-R94442018-1.pdf: 2398421 bytes, checksum: 7735c3ac65e0c68ddc8dcd027ee11ef1 (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 口試委員會審定書……………………………………………………i
誌謝……………………………………………………………………ii 中文摘要………………………………………………………………iv 英文摘要………………………………………………………………v 英文縮寫表……………………………………………………………vi 第一章 緒論…………………………………………………………1 第一節 基因之功能研究發展與應用………………………………1 1.1 基因遞送之方式…………………………………………………1 1.2 非病型基因轉殖…………………………………………………2 1.3 物質輔助基因轉殖方法之發展…………………………………3 1.3.1 起源……………………………………………………………3 1.3.2 物質輔助基因轉殖……………………………………………3 1.3.3 聚合物輔助基因轉殖…………………………………………3 1.3.4 附著型聚合物輔助基因轉殖…………………………………5 1.3.5 固著型輔助基因轉殖…………………………………………6 1.4 界面基因轉殖……………………………………………………7 第二節 研究動機與目的……………………………………………8 第二章 實驗材料與方法……………………………………………10 第一節 實驗材料……………………………………………………10 1.1 質體基因…………………………………………………………10 1.2 藥品與材料………………………………………………………10 1.3 細胞株……………………………………………………………11 第二節 實驗方法……………………………………………………12 2.1 細胞培養材料製備………………………………………………12 2.2 細胞培養…………………………………………………………13 2.3 脂質製備…………………………………………………………14 2.4 脂質界面基因轉殖法……………………………………………15 2.5 界面基因轉殖效力分析…………………………………………16 第三章 結果…………………………………………………………17 第一節 脂質界面基因轉殖標準操作程序之建立…………………17 1.1 脂質界面基因轉殖搭配正價離子之最佳使用絛件分析………17 1.2 基因轉殖效率與細胞稠密度之關係分析………………………18 第二節 脂質界面基因轉殖之效力分析……………………………19 2.1 脂質塗佈界面之保存時效分析…………………………………19 2.2 質體基因與氯化鎂混合溶液之效力分析………………………19 2.3 界面脂質與混合溶液之保存時效分析…………………………20 2.4 乾燥後界面之基因轉殖效力分析………………………………20 第三節 脂質界面基因轉殖之應用…………………………………21 3.1 幹細胞之基因轉殖………………………………………………21 3.2 神經細胞之基因轉殖……………………………………………21 3.3 肝臟星狀細胞之基因轉殖………………………………………22 第四章 討論…………………………………………………………23 第一節 脂質界面基因轉殖離子使用量之探討……………………23 第二節 脂質界面基因轉殖之效力探討……………………………27 第三節 脂質界面基因轉殖之應用…………………………………30 第五章 參考文獻……………………………………………………32 第六章 圖表…………………………………………………………38 | |
| dc.language.iso | zh-TW | |
| dc.title | 脂質界面基因轉殖法之效力分析與應用 | zh_TW |
| dc.title | Efficacy analysis and application of lipid surfection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 莊榮輝(Rong-Huay Juang),許金玉(Jin-Yu Shiu) | |
| dc.subject.keyword | 脂質,界面基因轉殖,轉助基因轉殖,基因轉殖效率,穩定細胞系,氯化鈉,氯化鎂,硫酸鋅,肝臟星狀細胞,神經細胞,幹細胞, | zh_TW |
| dc.subject.keyword | lipid,surfection,substrate-mediated transfection,transfection efficiency,stable cell line,sodium chloride,magnesium chloride,zinc sulfate,hepatic stellate cells (HSC),neuron cells,stem cells, | en |
| dc.relation.page | 49 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-31 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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| ntu-96-1.pdf 目前未授權公開取用 | 2.34 MB | Adobe PDF |
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