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系統識別號 U0026-2908201209460700
論文名稱(中文) NIH/3T3纖維母細胞在直流和交流電場下移動行為的觀察
論文名稱(英文) The movement of NIH/3T3 cell in DC and AC electric field
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 100
學期 2
出版年 101
研究生(中文) 楊沁潔
研究生(英文) Ching-Chieh Yang
學號 N36994093
學位類別 碩士
語文別 中文
論文頁數 113頁
口試委員 指導教授-王翔郁
口試委員-謝之真
口試委員-胡晉嘉
中文關鍵字 電趨性  纖維母細胞 
英文關鍵字 galvanotaxis  fibroblast 
學科別分類
中文摘要 細胞電趨性的研究已有一百多年的歷史。不論是體內實驗(in vivo)或是體外實驗(in vitro)皆可觀察到細胞電趨性行為。在電場的作用下,細胞移動的方向依細胞種類不同而有所不同(趨向正極或是趨向負極),有些細胞則不受電場影響。進行體外電趨性實驗時使用微流體裝置的優點是裝置較易組裝、裝置成本低、細胞樣本需求量低、容易控制電場、產生的焦耳熱(Joule heat)可快速散失。然而,避免細胞溶液和電解產物接觸為實驗時須考量的問題之ㄧ。進行電趨性實驗時,需要長時間外加電場於實驗裝置上,因此在實驗的過程中可能會有毒性電解產物產生,造成細胞死亡。本研究利用光聚合性高分子 (poly diallyldimethylammonium chloride,PDADMAC)製作鹽橋來區隔細胞溶液和電解產物。此種高分子具有生物相容性,不會毒害細胞,且其固化的形狀及位置相當容易控制,因此非常適用於微流體實驗。本實驗利用96 wt%Diallyldimethylammonium chloride加入2 wt%2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone以及2 wt% N,N'-methylenebisacrylamide的混合溶液經UV光聚合固化鹽橋,在曝光時間為40秒時,可得到結構完整及表面光滑的鹽橋。
為了驗證本研究的微流體裝置是否可用於電趨性實驗,首先使用NIH/3T3細胞在此裝置內進行直流電場內的電趨性實驗。實驗所得到的結果和文獻上的資料相符:NIH/3T3細胞暴露於直流電場下會往負極移動,電場值由292mV/cm增加至3166mV/cm時,細胞平均移動方向性由0.52增加為0.79。目前電趨性實驗大多聚焦於直流電場下細胞行為的研究,交流電場下的細胞行為觀測相對較少。所以,本研究觀察了不同頻率下細胞移動的行為模式,發現:(1)電場相同時,低頻環境下細胞任意移動的比例較在高頻環境下高,(2)頻率相同時,電場越大,越多的細胞趨向電極移動。
英文摘要 Researches for cell galvanotaxis have been over one century. We can observe the movement guided by electric field no matter in vivo or in vitro. In electric fields, some cells moved toward specific direction (toward anode or cathode), while some cells moved randomly(cell did not influence by electric field).The advantages of using microfluidic devices to conduct galvanotaxis experiment are:(1)it is easy to fabricate the devices, (2)the cost for fabricating the devices is low, (3) tiny cell amount is required for the experiment, (4) it is easy to control the electric field in the device, (5) Joule heat can be removed rapidly. During the galvanotaxis experiment, we need to apply the electric field for a long time, and the electrodes may generate electrolytic products that are toxic and result in cell death. In this research, we used conductive polymer (poly diallyldimethylammonium chloride,PDADMAC) to fabricate on-chip salt bridge. This material is biocompatible and it is easy to control the shape and location of salt bridge in the microfluidic channel. For fabricating smooth and complete salt bridge, the compositions of the salt bridge we used was 96 wt% Diallyldimethylammonium chloride, 2 wt% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, and 2 wt% N,N'-methylenebisacrylamide, and the exposure time under UV light was 40s.
To make sure this microfluidic device can be used for galvanotaxis experiment, we first used NIH/3T3 cell to conduct galvanotaxis experiment in direct current (DC) electric field. The experiment results are similar to those in the reference:NIH/3T3 cell moved toward cathode in DC electric field. When electric field strength increased from 292mV/cm to 3166mV/cm, the average directedness increased from 0.52 to 0.79. Most galvanotaxis experiments focused on cell’s behavior in DC electric field, and the research using AC electric field is few. Therefore, we observed the behavior of cells under AC electric field with different frequencies and strengths. The results show that:(1)in the same electric field strength, the ratio of cells moved randomly was higher in low frequency than in high frequency, (2) with the same frequency, more cells moved toward electrode when the electric field strength increased.

論文目次 第一章緒論 1
1.1前言 1
1.2研究動機與方法 2
第二章文獻回顧 3
2.1細胞的電趨性 3
2.1.1上皮電位 3
2.1.1.1生理電場對於傷口癒合時的作用 4
2.1.1.2生理電場對於胚胎發育時的作用 5
2.1.1.3生理電場對神經細胞發展時的作用 5
2.1.2細胞在電場作用下的行為模式 6
2.1.3電場影響細胞的機制 8
2.1.4 細胞運動和細胞內物質的關係 10
2.1.5交流電場對細胞的影響 17
2.1.6改變電場方向對細胞移動的影響 20
2.1.7纖維母細胞 20
2.2微流體系統中的電趨性實驗 21
2.2.1軟蝕刻 21
2.2.2微流體系統中的電趨性實驗 22
2.3鹽橋 24
2.3.1利用洋菜膠製成鹽橋的電趨性實驗裝置 24
2.3.2利用導電性高分子製成鹽橋的電趨性實驗裝置 25
第三章實驗材料與方法 29
3.1實驗材料 29
3.1.1黃光顯影製程 29
3.1.2高分子翻模製程 30
3.1.3製作鹽橋藥品 30
3.1.4製作微電極藥品 30
3.1.5細胞培養 30
3.2實驗儀器 33
3.3微流道製作 34
3.3.1清洗晶圓 34
3.3.2塗佈光阻劑 35
3.3.3軟烤 35
3.3.4曝光 35
3.3.5曝後烤 36
3.3.6顯影 36
3.3.7硬烤 36
3.4PDMS翻模 36
3.5鹽橋製作 37
3.6微流道內的電場強度測試 39
3.6.1微電極製作 39
3.6.2微流道內的電場強度測試 41
3.7利用UV光光度計及拉曼測試玻片上的高分子殘留 42
3.8細胞培養方法 43
3.8.1冷凍細胞活化 43
3.8.2細胞繼代培養 43
3.9電趨性實驗 44
3.10分析方法 45
3.10.1直流電電場中細胞移動分析方法 45
3.10.2交流電電場中細胞移動分析方法 46
3.10.3 細胞移動平均方向性 46
第四章結果與討論 47
4.1晶片上鹽橋的製造 47
4.1.1 98%P,1% I,1% L 47
4.1.2 97%P,1.5%I,1.5%L 48
4.1.3 97%P,1%I,2% L 50
4.1.4 96%P,2%I,2%L 50
4.1.5結論 52
4.2表面高分子殘留測試 52
4.2.1吸收度測試 53
4.2.2拉曼(Raman)光譜測試 53
4.3鹽橋間的電場強度 54
4.4直流電電場下的電趨性實驗 56
4.4.1無電場作用下細胞移動的趨勢 59
4.4.2施加電場後細胞移動的趨勢 61
4.4.3結論 71
4.5交流電電場下的電趨性實驗 73
4.5.1頻率10Hz 73
4.5.2頻率10KHz 84
4.5.3頻率10MHz 95
4.5.4 結論 107
第五章結論與未來展望 108
5.1結論 108
5.2未來展望 108
第六章參考文獻 109

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