經酸酐修飾的藍藻蛋白之細胞毒性探討｜國立臺灣科技大學博碩士論文系統

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研究生：	蔡依廷 I-Ting Tsai
論文名稱：	經酸酐修飾的藍藻蛋白之細胞毒性探討 Cellular toxicity of cyanophycin modified with anhydride
指導教授：	曾文祺 Wen-Chi Tseng
口試委員:	陳燿騰 Yaw-Terng Chern 方翠筠 Tsuei-Yun Fang 孫幸宜 Shing-Yi Suen
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2012
畢業學年度：	100
語文別：	中文
論文頁數：	80
中文關鍵詞：	酸酐修飾的藍藻蛋白
外文關鍵詞：	cyanophycin modified with anhydride
相關次數：	點閱：180 下載：0
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上一筆

藍藻蛋白為非核醣體合成蛋白質，由aspartic acid、arginine、 lysine三種胺基酸所組成；而藍藻蛋白上的lysine上包含了胺基，容易進行化學修飾。本研究是將不同種類的有機酸酐與藍藻蛋白上的胺基進行醯化反應，期望拓展藍藻蛋白的應用。
醯化藍藻蛋白，可利用薄膜色層分析(TLC)、液態超導核磁共振儀(NMR)與傅立葉轉換紅外線光譜儀(FTIR)確定其結構，並經由熱重/熱示差分析儀(TGA)觀察其物性變化。
透過DNA電泳可以看出對DNA的包覆能力CGP-octanoic anhydride＞CGP-acetic anhydride＞CGP-myristic anhydride，其中CGP-octanoic anhydride可完全包覆DNA，可利用此結果進一步地去探討對動物細胞的轉染效果。
本實驗利用CHO cell對醯化藍藻蛋白進行毒性測試，經過48小時的細胞培養，發現相較於控制組，培養液中加入醯化藍藻蛋白水溶液，可提升細胞存活率，其中在低濃度(100 μg/mL)以下效果較為明顯；對於Raw 264.7進行免疫測試中的蛋白質含量測定亦是同樣的結果。
結果得知，藍藻蛋白經由醯化反應，有助於細胞生長，所以醯化藍藻蛋白若能進一步的改質，除了有機會可以當作藥物載體，也期望能應用在生醫材料方面的領域中，增加藍藻蛋白的應用價值。

Cyanophycin (CGP) is a non-ribosomal synthesis protein which is composed aspartic acid, arginine and lysine. The lysine molecular, which contains primary amine, can allow chemical modifications with anhydride grafting. In this study, we use different types of anhydride and attempt to expand the applications of the cyanophycin.
For characterization of structure, we used FTIR to analyze the functional groups of cyanophycin modified with anhydride and TGA to detect the thermal stability.
For DNA/polymer complex tests, the encapsulation ability follows the order of CGP-octanoic anhydride > CGP-acetic anhydride > CGP-myristic anhydride with DNA electrophoresis. Especially CGP-octanoic anhydride can completely encapsulate DNA, so we can use this result to explore the transfection efficiency of animal cells.
For biocompatibility, the cytotoxicity tests of cyanophycin modified with anhydride uses MTT method after cells incubate for 48 hours. We can find that cyanophycin modified with anhydride comparing to controlling improve apparently, especially in low concentration. In addition, the effect of the protein test in the immune response is also the same result for Raw 264.7.
To sum up, the cyanophycin modified with anhydride provides a better condition for cell growth. If the product of cyanophycin modified with anhydride can further modification, can be drug carriers. Also we hope to have the opportunity to look forward to the application in biomaterials area.

中文摘要.......................................................................	I
ABSTRACT...............................................................	II
致謝...................................................................	III
目錄...................................................................	IV
圖目錄	...............................................................VIII
第一章 緒論....................................................................	1
第二章 文獻回顧................................................................	2
1 藍藻蛋白簡介...............................................................	2
1.1 藍藻蛋白的特性...........................................................	3
1.2 基因重組藍藻蛋白	........................................................4
1.3藍藻蛋白之應用............................................................	5
2 醯化反應..................................................................	7
3免疫反應	...............................................................10
3.1 一氧化氮.........................................................	11
3.2 脂多醣...........................................................	12
3.3 NOx release定量測試..............................................	14
3.4 Raw 264.7 巨噬細胞...............................................	16
4生醫材料的生物性質..................................................	17
4.1 MTT毒性測試......................................................	18
第三章 實驗部分........................................................	20
1實驗材料............................................................	20
1.1菌株..............................................................	20
1.2細胞株............................................................	20
1.3細胞培養液	........................................................20
1.4抗生素............................................................	20
1.5其他..............................................................	21
2實驗藥品............................................................	21
3實驗儀器............................................................	23
4溶液配製............................................................	24
5實驗步驟............................................................	27
5.1藍藻蛋白的生.....................................................	27
5.2藍藻蛋白醯化反應之製備............................................	31
5.3醯化藍藻蛋白對DNA包覆測試.........................................	32
5.4 細胞培養與轉染效率測定...........................................	33
5.5 醯化藍藻蛋白對Raw264.7相容性測試.................................	34
5.6 細胞培養與繼代...................................................	35
6物性測試............................................................	37
6.1熱重/熱示差分析儀（TGA）測試......................................	37
6.2傅立葉轉換紅外線光譜儀( FTIR )鑑定................................	37
6.3 核磁共振光譜儀(NMR)鑑定..........................................	37
第四章 結果與討論....................................................	39
1傅立葉轉換紅外線光譜儀(FTIR)鑑定結果................................	39
1.1 CGP-g-acetic anhydride FTIR鑑定..................................	39
1.2 CGP-g-octanoic anhydride FTIR鑑定................................	40
1.3 CGP-g-myristic anhydride FTIR鑑定................................	40
2	NMR數據分析結果	................................................41
2.1 CGP-g-acetic anhydride NMR鑑定..................................	41
2.2 CGP-g-octanoic anhydride NMR鑑定.................................	41
2.3 CGP-g-myristic anhydride NMR鑑定.................................	41
3熱重/熱示差分析儀結果分析..........................................	42
3.1醯化藍藻蛋白TGA結果分析...........................................	42
4 溶解度測試.........................................................	43
5 DNA電泳結果分析....................................................	44
5.1 CGP-g-acetic anhydride 電泳結果..................................	44
5.2 CGP-g-octanoic anhydride 電泳結果	................................44
5.3 CGP-g-myristic anhydride 電泳結果.................................45
6	CHO cell 毒性測試...............................................45
6.1 CGP-g-acetic anhydride對CHO cell生長影響..........................45
6.2 CGP-g-octanoic anhydride對CHO cell生長影響........................46
6.3  CGP-g-myristic anhydride對CHO cell生長影響.......................46
7 LPS誘導Raw 264.7....................................................47
8 Raw 264.7免疫測試...................................................48
第五章 結論.............................................................50
參考文獻................................................................52
圖表....................................................................55

                                

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全文公開日期 2027/07/27 (國家圖書館：臺灣博碩士論文系統)

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