臺灣大學: 高分子科學與工程學研究所楊台鴻陳頤欣Chen, Yi-HsinYi-HsinChen2013-03-292018-06-292013-03-292018-06-292012http://ntur.lib.ntu.edu.tw//handle/246246/254434幾丁聚醣 (Chitosan)，地球上第二大天然多醣體幾丁質(Chitin)之主要衍伸物，因天性有高度的生物相容性，生物可裂解性, 低生物毒性等優勢, 於生物醫學方面有諸多應用。在結構上，以葡萄醣胺及甲基葡萄醣胺為主要單體，隨機交錯排列聚合而組成。由於葡萄醣胺上之初級胺結構，幾丁聚醣在應用上，被視為一個正電高分子，具有環境酸鹼應答(pH-responsive)之特性，並被廣泛地應用在諸如藥物釋放，組織工程，醫學材料等相關領域。然而，這種酸鹼應答的特性從來沒被應用或討論於一個細胞培養系統。本篇論文即應用幾丁聚醣天性上特殊的酸鹼應答特性，開發出一個藉由調控環境酸鹼值而控制細胞與幾丁聚醣交互作用的細胞培養平台。我們發現，幾丁聚醣於哺乳類細胞培養環境具有酸鹼應答之特性，這個應答特性可以藉由很小的環境酸鹼值改變，進而調控細胞貼附於幾丁聚醣上的行為；並且，這個特性可以被應用於脫附回收培養在幾丁聚醣上的細胞，不必額外添加酵素或藥劑，省去繁複及對細胞有害的步驟。於本論文中，我們並闡述細胞之貼附，纖維連結蛋白之吸附，與培養基酸鹼值三者之間的關係。進一步，我們發現細胞對於材料之酸鹼應答有不同的脫附反應，而不同的脫附速率與其細胞貼附的形狀有關。根據這個特性，我們開發出一個經濟，快速，操作簡便，又可廣泛應用的細胞篩選平台。利用人工混和不同脫附速率的細胞，我們驗證了這個平台對於細胞篩選的可用性。另外，本論文也驗證了，這個平台對於初代腎小管上皮細胞培養亦有其重要性。更進一步地，我們討論了細胞骨架在幾丁聚醣上之酸鹼應答脫附中的重要性，以人類纖維母細胞Hs68為例，說明了Rho/ROCK相關之細胞骨架活動在這個脫附行為中所扮演的角色。另一方面，我們試圖尋找限制圓形細胞留在幾丁聚醣上，不因酸鹼應答脫附的原因。以人類角質上皮細胞株 HaCaT為例，我們發現限制HaCaT因酸鹼應答脫附的原因，和β4-integrin及其配體之作用有關。最後，我們將進一步討論這個平台在未來的開發及其應用性。Chitosan, the major derived from second abundant nature polymer chitin, has many biomedical applications because of its biocompatibility, biodegradability, and low toxicity. Structurally, it is a nature cationic polysaccharide composed randomly distribution of N-glucosamine and N-acetyl-glucosamine groups. The presence of primary amine of glucosamine residue makes chitosan a pH-responsive polycation and has potential to apply in pH-responsive aspect, such as drug delivery, tissue-engineering, or related medical device. However, this pH-responsibility has never been applied and discussed for a cell culture system. In this thesis, we developed a culture system based on the pH-responsive nature of chitosan which can control the cell-substrate interaction by precise adjustment of medium pH. We discovered that pH-responsive chitosan was able to control cell behavior in response to small changes in environmental pH, which was at useful pH suitable for recovering cultured cells without additional enzymatic treatment and extensive washing steps. The relationship of cell attachment/detachment, serum fibronectin adsorption, and medium pH was illustrated. Furthermore, a time and cost effective, simple-operative, and wide-extensive platform based on chitosan was developed, based on the different cell detachment ratio from chitosan when raising medium pH. The different detachment ratio was found to be correlated with cell shape. By artificial mixing different detachment ratio of cells, the application of this system was revealed. The importance of this system for primary renal proximal tubular epithelial culture was also demonstrated. Moreover, the role of intracellular cytoskeleton in this pH-induced detachment from chitosan surface was further investigated. The importance of Rho/ROCK-related cytoskeletal action was illustrated. In addition, the reason of HaCaT restricted by this pH-induced detachment on chitosan was investigated. The role of β4-integrin and its ligand in the restriction of pH-induced detachment from chitosan was discussed. Finally, the future perspective of this pH-responsive chitosan culture system was demonstrated.116278168 bytesapplication/pdfen-US幾丁聚醣纖維黏附蛋白細胞貼附酸鹼應答材料細胞培養系統細胞篩選Chitosanfibronectincell adhesionpH-responsive materialscell culture systemcell selection.thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/254434/1/ntu-101-F93549004-1.pdf