楊台鴻臺灣大學:醫學工程學研究所張馨淳Chang, Hsin-ChunHsin-ChunChang2010-05-182018-06-292010-05-182018-06-292009U0001-1301200900402100http://ntur.lib.ntu.edu.tw//handle/246246/183692聚乙烯乙烯醇(EVAL)是一種近年來常利用的高分子材料。本實驗室中已經證實，將纖維母細胞培養在莫耳比例不同的聚乙烯乙烯醇薄膜表面，經過一段時間後，細胞在老化相關的基因、生長曲線、細胞形態都產生明顯的差異。但缺點是此實驗因為正常體細胞，分裂速度較慢，必須長時間才能完成實驗，並且因為長期培養，還可能要承擔污染或是細胞狀況不穩定的風險。因此在本實驗中，主要目的是希望改善研究老化議題時需長期培養細胞的缺點，建立一個有效率地測試材料對細胞是否造成老化影響的平台。 在這裡使用鼻咽癌的細胞株HONE-1以及非小細胞肺癌細胞株H1299兩種癌細胞作為老化現象的觀測細胞，主要是因為癌細胞分裂次數較快，而且在探討其因為外力造成老化的前驅性老化時，可避免正常體細胞會有的複製性老化現象。其中，H1299缺少了老化常有過度表現的p53基因，在此也可探討p53基因的有無，是否對細胞的老化造成影響。實驗中選用聚乙烯莫耳比例27%(EVAL27)及44%（EVAL44）以及商業用的組織培養盤（TCPS）作為實驗中的三種不同實驗材料。 實驗分為兩部分：第一部分是探討將細胞培養在不同材料上是否會造成老化表現的差異。其中包括培養在高分子材料上但不施予其他應力、同時施予5%乙醇二小時與高分子材料兩種應力、或是先使用乙醇處理後在培養在不同材料上等三種實驗流程。第二部分探討施予不同濃度乙醇處理後的細胞，是否會在三種材料上出現生長的差異。 實驗顯示：若是僅將細胞培養在不同的材料上，細胞僅會有生長速度不同的現象，並不會對細胞造成任何老化的效果。而若是繼代培養加入5%乙醇的浸泡，卻使得細胞因為乙醇而快速造成老化，觀察不出材料間是否有不同的差異，而調整的浸泡乙醇的次數，卻沒有改善上述的情形。不同濃度乙醇的處理顯示在某些濃度的乙醇處理下，細胞的生長速度會產生差異，TCPS表現的細胞分裂速度較快，但其他兩種材料間差異不大。因此，實驗顯示這樣設計的實驗平台效果有限，三種不同的材料並不會對細胞株造成老化的影響，而僅有生長速度略微不同的差異而已。而乙醇造成的傷害太大，也無法比較三種材料間的差異。 由實驗的結果推斷：這三種材料對於細胞株的影響能力有限，僅僅在細胞的分裂速度上會有些微的差距。而加上浸泡數次乙醇或是不同濃度乙醇處理，對於細胞內累積過多外來應力，造成細胞株老化、死亡，但是卻無法分辨三種材料間是否對細胞有不同的影響。因此這個平台設計的效果並不如預期好，可以看出材料影響細胞的生長速度，但無法有效改善探討老化時長時間培養的缺點。Poly (ethylene-co-vinyl alcohol) (EVAL) is a copolymer which has been widely used as biomaterials recently. In our laboratory, it is proved that primary skin dermal fibroblasts cultured on EVAL biomembranes have significant influences on cell morphology, growth curve, and gene expression associated with senescence. However, the recent process of studying senescence phenomenon has some major shortcomings such as slow-rating division, long time for cultivation, and risks of pollution or poor cell conditions occasionally. In this research, the main purpose is to set up a quick-acting method, which can greatly shorten the cultivation time. Thus, the senescent effects by different biomembranes could be examined more effectively. Here, the Nasopharyngeal Carcinoma (NPC) cell line, HONE-1, and non-small cell lung carcinoma (NSCLC) cell line, H1299, are used as experimental cell to observe the behaviors of senescence. In this manner, the replicative senescence is avoided due to the rapid cleavage rate of the cell line. On the other hand, because of the lack of p53 gene, the cell line H1299 may go to senescence in another pathway different from that originally involved in p53 gene. In the point of view, the effect of gene p53 on cell senescence can also be discussed. Besides, in the experiment, the cell is cultured on three types of surface; they are EVAL44 (EVAL which composed of 44 mol% hydrophobic ethylene segment), EVAL27 (EVAL which composed of 27 mol% hydrophobic ethylene segment), and TCPS (tissue culture polystyrene plates), respectively. The experimental procedure is classified into three modes. One mode is that the cells are cultured on the three types of biomembrane surefaces for a long time to distinguish the differences among them. Another one is that the cells are cultured on the three biomembrane surfaces and exposed to ethanol at the same time. The other mode is that the cells are cultured on the three biomembrane surfaces after exposure to ethanol for several times. After that, the senescence effects on the cell line by the different biomembranes were observed. As shown in the results, however, the three types of surface have limited influence on the cancer cell line. If the cells were treated with ethanol, they may go to the senescent stage. Otherwise, if ethanol treatment was cancelled, the difference in division rate can hardly be observed, but the cells didn’t go to senescence. Therefore, the designed process is not as satisfactory as expected, for its inability to improve the long-time consuming problem.目錄謝……………………………………………………I要…………………………………………………IIIbstract………………………………………………V錄…………………………………………………VII目錄………………………………………………IX一章 序論…………………………………………1二章 文獻回顧……………………………………3一節 細胞老化……………………………………3二節 細胞培養……………………………………9三節 生醫材料……………………………………11四節 材料對細胞的影響…………………………12三章 材料與方法…………………………………14一節 實驗藥品……………………………………14二節 實驗儀器……………………………………16三節 藥品配製……………………………………17四節 實驗方法……………………………………19四章 實驗設計……………………………………24一節 實驗目的……………………………………24二節 參考的文獻流程……………………………24三節 實驗設計流程………………………………24四節 流程圖………………………………………26五章 結果與討論…………………………………27一節 細胞活性與細胞毒性試驗…………………27二節 細胞貼附、脫附能力………………………28三節 生長曲線……………………………………31四節 細胞形態……………………………………38五節 senescence-associated β-galactosidase 染色情形…47六節 蛋白質表現…………………………………52七節 細胞倍增時間、細胞活性、細胞毒性與乙醇的關係……66八節 綜合討論……………………………………68六章 結論…………………………………………71參考文獻……………………………………………72附錄…………………………………………………81application/pdf4157099 bytesapplication/pdfen-US細胞老化高分子生物薄膜細胞株聚乙烯乙烯醇共聚物乙醇cellular senescencepolymer biomembranescell linepoly(vinyl alcohol-co-ethylene)ethanol[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/183692/1/ntu-98-R95548061-1.pdf