林峯輝Lin, Feng-Huei臺灣大學:醫學工程學研究所黃建元Huang, Jian-YuanJian-YuanHuang2010-05-182018-06-292010-05-182018-06-292009U0001-1308200910415700http://ntur.lib.ntu.edu.tw//handle/246246/183701遺傳性的疾病造成某些病人無法藉由自體產生必要蛋白質，往往需透過體外給予醫藥蛋白方能維持正常的生理機能。不過目前許多醫藥蛋白價格昂貴且產量少萃取不易。使用微生物(如E.coli、酵母菌等)將外源基因嵌入進而培養萃取特用蛋白，該方法不能大量生產且某些蛋白質無法修飾直接供人體利用，須另外在處理，其過程繁瑣。所以學者利用高等哺乳類動物，做為基因轉殖動物以生產大量的醫藥蛋白，以哺乳類動物做為生物反應器生產醫藥蛋白為近年學者研究的重心。 而基因轉殖雞的研究上不僅培養子代的速率較哺乳類動物快，且雞蛋蛋白的成分較哺乳類動物的乳液單純，在萃取上便捷許多。所以利用基因轉殖雞做為製造蛋白質的生物反應器有許多的優勢在。 本研究利用正電荷改質之明膠/磷酸鈣奈米微粒做為基因載體，其粒徑分佈不超過1μm，有利於細胞吞噬。由電泳測試知該微粒能有效保護與包覆DNA能力，具有做為良好基因載體的優勢。在生物相容性測試中，正電荷改質之明膠/磷酸鈣奈米微粒比起市售之LipofectamineTM2000更為安全，且可達百分之四十的轉染效率。在雞胚轉染實驗中，可由被轉染第四天之雞胚利用共軛焦顯微鏡觀察到綠色螢光，證明正電荷改質之明膠/磷酸鈣奈米微粒具有良好的生物相容性與轉染能力，具有生產基因轉殖雞的潛力。Due to the hereditary disease, some specific proteins cannot be synthesized for the maintenance of physiological function. The pharmaceutical proteins, prepared in recombinant bacteria, play an important role in the medical engineering. However, most of pharmaceutical proteins are expensive and the production rate is low. Additionally, the proteins prepared from bacteria are inactive or unstable in vivo since they cannot be matured by post-translational modifications. Therefore, the transgenic animal system is a promising bioreactor to produce active proteins having an appropriate biochemical structure. he advantages of using transgenic chickens as bioreactors are the short incubation time, the high production rate, the sterilized contents of the egg, and the stable proteins in egg. Moreover, the contents of the proteins extracted from the hen’s egg compared with the milk from mammals are simpler. Therefore, the transgenic chickens have the potential to be an ideal bioreactor for the production of specific proteins in the future.n this study, the gelatin/calcium phosphate nanoparticles after the cationization can be used as gene carriers applied in the transgenic chickens. Because the particle size is smaller than 1um, it has more possibility for the cell uptake. Using electrophoresis enables to evaluate the DNA loading efficiency, and the result shows that the nanoparticles have ability to carry DNA and protect the DNA as well. The nanoparticles also have higher biocompatibility than the commercial product, LipofecamineTM2000. The transfection efficiency of the cationizated gelatin/CaP nanoparticles achieves at least 40%. In the animal study, it is found that the embryo emits green fluorescence protein observed by the fluorescence microscope after the fourth day of the experiment, so the cationizated gelatin/CaP nanoparticles have potential applied in transgenic chicken.中文摘要 I文摘要 II錄 IV目錄 VII目錄 IX一章 前言 1-1 醫藥蛋白 1-2 基因轉殖動物 2-3 基因轉殖雞 4-4 基因轉染技術 5-4-1 顯微注射法 5-4-2 胚胎幹細胞植入法 6-4-3 反轉錄病毒感染法 7-5 基因遞送系統 8-5-1 病毒載體 8-5-2 非病毒載體 9-6 研究目的 12二章 理論基礎 13-1 基因載體進入細胞內的機制 13-2 明膠 15-3 明膠奈米微粒發展 16-4 磷酸鈣在基因載體上的發展 18三章 實驗方法 19-1 實驗儀器 19-2 實驗藥品 21-3 實驗流程及材料製備 23-3-1 實驗架構 23-3-2 明膠奈米微粒製備 24-3-3 明膠/磷酸鈣 - DNA微粒製備 26-4 材料分析、生物相容性測試與體內、體外實驗 28-4-1 質體大量萃取 28-4-2 粒徑與界面電位測量 29-4-4 穿透式電子顯微鏡分析 30-4-5 掃描式電子顯微鏡分析 30-4-6 原子力顯微鏡分析 31-4-7 DNA包覆率測試 31-4-8 電泳分析 32-4-9 WST – 1細胞活性測試 32-4-10 LDH細胞毒性測試 33-4-11 體外細胞轉染實驗 34-4-12 動物實驗 35四章 結果與討論 36-1 明膠/磷酸鈣奈米微粒之製備結果 36-1-1 DLS粒徑分析 36-1-2 傅立葉轉換紅外線光譜儀(FT-IR) 38-1-2 掃描式電子顯微鏡分析 39-1-3 原子力顯微鏡分析 40-1-4 穿透式電子顯微鏡分析 42-1-5 表面電位分析 45-2 包覆效率測試 47-3 奈米微粒保護核酸抵抗限制酶之電泳分析 48-3 生物相容性測試 50-4 體外細胞轉染實驗 52-5 動物實驗 59五章 結論 62考文獻 63application/pdf12436914 bytesapplication/pdfen-US基因載體基因轉殖雞明膠磷酸鈣gene carriertransgenic chickengelatincalcium phosphatethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/183701/1/ntu-98-R96548015-1.pdf