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系統識別號 U0026-0812200915390581
論文名稱(中文) 以阻抗分析系統評估維生素C處理下所影響之細胞修復行為
論文名稱(英文) Using Impedance Sensing System to Evaluate the Effect of Wound Healing Behavior on Cells Treated by Vitamin C
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 97
學期 2
出版年 98
研究生(中文) 李孝貞
研究生(英文) Hsiao-chen Lee
電子信箱 s640125@yahoo.com.tw
學號 p8696118
學位類別 碩士
語文別 中文
論文頁數 54頁
口試委員 指導教授-張憲彰
口試委員-王德華
口試委員-沈孟儒
口試委員-丁信智
召集委員-張炳華
中文關鍵字 傷口修復  維他命C  電傷害  阻抗分析 
英文關鍵字 Wound Healing  Electric Impedance Sensing System  Electric Injury  Vitamin C 
學科別分類
中文摘要 生物學上所謂的受傷(wound),是指正常的組織結構或功能受到破壞;而癒合(healing)則是指重建完整解剖構造及功能所經過的一連串複雜動態過程,此過程中有許多因子可促進或抑制此過程,如:維他命C即是促進的因子。實際上,不論組織或細胞,每時每刻都面臨著外來的刺激或傷害;尤以細胞層級的傷害,更是在組織發生變化前就開始發生。故若能對細胞的傷害與癒合進行即時的分析,則對瞭解受傷機轉和研發藥物都有相當的幫助。Scracth wound assay和MTT assay是生物學上常被用來研究細胞層傷害癒合的標準方法,但卻各有很難量化傷口癒合的動態過程,與僅能做各個特定時間點的檢測之缺點。另一個可供選擇則是阻抗量測(EIS)法,其優點為可即時監測細胞變化、易於進行量化分析,亦可依據實驗需求設計不同的電極進行檢測。本研究是將纖維母細胞(L929)培養在以微機電技術製作出的微小城垛型指插電極(IMP)上,利用EIS記錄細胞貼附、受到電傷害的變化和癒合過程,以及維他命C對此過程的影響;同時進行連續攝影,並與MTT assay比較分析。細胞在受到直流電傷害後,在正極上的細胞會因細胞膜穿孔導致細胞產生先腫脹後死亡的變化,然後由旁邊的細胞進行傷口修復。研究結果顯示,此變化在EIS上可即時且連續監測,且其修復趨勢與MTT assay相同。此外,在阻抗量測上,可發現細胞電傷後立即給予100 M維他命C可加速細胞的修復;雖在電傷前給予維他命C無法減少細胞受損,卻同樣可加速細胞的修復。由此可知,EIS是即時且連續監測貼附型細胞變化的有效方法;而IMP則是能量測阻抗變化,亦能造成細胞傷害或死亡的微小電極。此微小電極配合阻抗量測系統不僅可即時研究電傷害對細胞的影響和藥物對電傷修復過程的影響,且可迅速且即時分析細胞產生變化的時間點;並在變化的時間點上,再輔以生化技術進行進一步的分析。對研究傷害癒合和研發藥物而言,是相當有利的工具。
英文摘要 The healing of tissue wound is a cascade of complex, dynamic and multi-cellular process. Many factors control and coordinate this process, including vitamin C. Vitamin C, L-ascorbic acid, is an essential nutrient for humans, an effective antioxidant, an enzyme cofactor for the biosynthesis of collagen, and an electron donor for many enzymes. The dynamic change after cell injury by electric forces, cellular healing process of electric injury, and the role of vitamin C in the healing process are still not clear. Biologically scratch wound assay and MTT assay are the standard methods to evaluate the cellular healing, but both of them can’t detect the dynamic change of healing process. Electrical impedance sensing system (EIS) is an alternative to detect cellular healing process in real-time. In this thesis, we used EIS to monitor the healing process of L929 fibroblasts injured by 30 A direct current (DC) and the effect of 100 M vitamin C added before and after electric injury on interdigitated microelectrode plates (IMP). We found that the size of damaged cells increased first then these cells died and disappeared from IMP gradually and healing process started at the same time. This was compatible with the result of MTT assay. Treated cells with vitamin C after electric injury decreased damage. Besides, the damage in the group of adding vitamin C before electric injury didn’t decrease, but the healing rate is improved.
論文目次 目 錄 IV
摘 要 VII
Abstract VIII
誌 謝 IX
圖 目 錄 X
表 目 錄 XIV
第一章 緒論 1
1-1 傷口的成因 2
1-1-1 內因性因子(intrinsic factors) 2
1-1-2 外因性因子(extrinsic factors) 4
1-2 細胞傷害機轉 5
1-3 傷口癒合之機轉 8
1-3-1 發炎期(inflammatory phase) 8
1-3-2 增生期(proliferative or fibroblastic phase) 9
1-3-3 重塑期(remodeling phase) 10
1-4 臨床上對傷口的處置 11
1-5 維生素C對傷口癒合之影響 12
1-5-1 維他命C概論 12
1-5-2 維生素C的生理功能 13
1-5-3 維他命C與傷口癒合 14
1-6 評估傷口癒合的方法 16
1-6-1 Scratch wound assay 16
1-6-2 MTT assay 17
1-6-3 Electric impedance sensing system 18
1-7 研究動機 23
1-8 論文之研究架構 24
第二章 研究方法 25
2-1 實驗設備及藥劑 25
2-1-1 HP 4194A Impedance/Gain-Phase Analyzer 25
2-1-2 使用LabVIEW收集data 25
2-1-3 Live cell instrument system and microscope 26
2-1-4 Keithley 2611 System SourceMeter 26
2-1-5 ELISA reader 26
2-1-6 細胞培養的材料與實驗藥劑的泡製 27
2-2 晶片和PDMS culture well製作 28
2-2-1 晶片製作 28
2-2-2 PDMS culture well製作 29
2-3 細胞培養 30
2-3-1 晶片上的細胞培養 30
2-4 實驗設計 31
2-4-1 細胞貼附之阻抗變化 31
2-4-2 細胞傷害模式 31
2-4-3 維他命C對細胞修復的影響 34
第三章 結果與討論 35
3-1 細胞貼附之阻抗變化 35
3-1-2 分析頻率的選擇 37
3-1-3 細胞貼附的阻抗變化 39
3-2 細胞修復的阻抗變化 42
3-2-1 細胞傷害模式 42
3-2-2 細胞修復的阻抗變化 43
3-3 維他命C對細胞修復的影響 46
3-3-1 維他命C對細胞修復的影響 46
3-3-2 維他命C的濃度對細胞修復的影響 47
3-3-3 維他命C的給予時間對細胞修復的影響 48
第四章 結論 49
第五章 未來展望 50
5-1 晶片設計 50
5-2 阻抗系統之延伸應用 50
參考文獻 51
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