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系統識別號 U0026-2008201920473700
論文名稱(中文) 探討非熱微電漿對黑色素瘤細胞及纖維母細胞共培養系統之影響
論文名稱(英文) Study of the Non-Thermal Micro Plasma on the Co-culture of Melanoma Cells and Fibroblasts
校院名稱 成功大學
系所名稱(中) 材料科學及工程學系
系所名稱(英) Department of Materials Science and Engineering
學年度 107
學期 2
出版年 108
研究生(中文) 高禎志
研究生(英文) Chen-Chih Kao
學號 N56061420
學位類別 碩士
語文別 中文
論文頁數 68頁
口試委員 指導教授-廖峻德
共同指導教授-劉浩志
共同指導教授-王士豪
口試委員-邵佩琳
口試委員-王德華
中文關鍵字 非熱微電漿  抑制癌細胞生長  反應性氧物種  共培養  皮膚黑色素瘤  纖維母細胞 
英文關鍵字 non-thermal plasma  reactive oxygen species  co-culture system  melanoma cell  fibroblasts 
學科別分類
中文摘要 電漿是將部分氣體離子化的物質第四態,近年來許多研究已顯示其在抑制癌細胞生長之重要性,以往非熱微電漿(NTP)主要是透過直接照射於癌細胞或腫瘤來進行抑制,不過由於NTP 照射的面積及深度有限,因此對於皮膚表層尚未發生轉移的腫瘤抑制較有效。而目前關於NTP 抑制癌細胞的文獻大部分針對單培養系統來進行,本研究為了以更貼近人體腫瘤微環境去探討NTP 對癌細胞之研究,因此採用皮膚癌細胞中惡性度且致死率最高的黑色素癌細胞B16-F10 及其周圍的纖維母細胞L929 進行共培養並探討NTP 對此共培養系統之影響。首先透過電漿溫度測量及OES 光譜分析決定本實驗電漿機台He 流量參數為1.6 standard L/min (SLM),ROS 濃度測量分單培養及共培養系統比較,單培養B16F-10 吸收ROS 效率比單培養L929 高3-10%;而在共培養系統中,NTP 似乎會促進細胞分泌更多的ROS 擴散至細胞外來維持環境的ROS 平衡,因此測量到的ROS 濃度有提升的趨勢。第二階段將B16-F10 及L929 分別以單培養及共培養後,觀察兩培養系統中的生長差異,發現共培養系統中的B16-F10 生長速率比單培養來得快,顯示出共培養環境中的生長因子可能促進B16-F10 的生長;而180 s 電漿處理雖然可抑制共培養B16-F10 的存活率至38%,不過也造成共培養L929 僅剩54%的存活率。最後為了提升低劑量電漿之效果,比較NTP 進行單次及二次處理後的抑制癌細胞生長之效果,結果顯示在相同總電漿處理時間下,二次處理相對單次處理造成癌細胞凋亡的比例提升了20 %左右,顯示出低劑量電漿確實可透過處理多次之方法來提升其效果。透過本研究的共培養實驗可以更清楚影響癌細胞生長的可能因子,未來有機會可以將NTP 的應用延伸至影響細胞交互作用間的細胞激素,進而達到更有效抑制癌細胞之方法。
英文摘要 In this study, the co-culture system of melanoma cell B16-F10 and fibroblasts L929 was used to investigate the selective effect of non-thermal micro plasma (NTP). At first, to find the best helium flow rate for experiment, the plasma temperature and generated species intensity were measured under different He flow rate, and 1.6 SLM of He was proper for the further experiment. The cell viability assay was separated to mono- and co-culture system to see the cell-cell interaction in co-culture system after NTP treatment. The results showed that co-cultured B16-F10 was more resistant to NTP compared with mono-cultured B16-F10, and it could be due to the cytokines release in the co-culture system. In general, NTP selectively induce B16-F10 rather than L929 in both culture systems for low dose plasma (30-90 s); however, both cells in co-culture system encountered large toxicity for high dose plasma (180 s), leading to around 50-60 % of cell death. Therefore, the double low dose treatment was replaced with single high dose treatment to keep higher living normal cell rate and inhibit cancer cell growth as usual. The results showed that in the same total treatment time, double plasma treatment enhanced around 20 % of B16-F10 apoptosis compared with single treatment, and leaving around 80-90 % of L929 alive.
論文目次 摘要................................Ⅰ
SUMMARY.............................Ⅱ
誌謝................................XII
表目錄............................... XVI
圖目錄................................ XVII
第一章 緒論..................................... 1
1.1 前言..................................... 1
1.2 研究動機................................... 4
1.3 研究目的................................... 4
第二章 文獻回顧與理論基礎............................ 5
2.1 造成細胞凋亡的電漿影響因子...................... 5
2.1.1 ROS 及RNS ............................... 5
2.1.2 UV 光.................................. 6
2.1.3 培養基pH 改變............................. 7
2.2 電漿應用於in vitro 與 in vivo 之研究................... 8
2.2.1 不同電漿系統及參數應用於in vitro 之研究............. 8
2.2.2 電漿對co-culture system 之影響................... 12
2.2.3 電漿應用於in vivo 之研究...................... 13
2.3 細胞外ROS 持續作用時間........................ 14
2.4 分次治療(fractionation)對於癌症研究的重要性.............. 17
2.4.1 分次治療應用於放射性療程之理論.................. 17
2.4.2 類似分次治療之電漿應用...................... 18
2.5 腫瘤微環境對癌細胞影響之研究.................... 19
2.5.1 活化的纖維母細胞促進了癌細胞的生長............... 19
2.5.2 HGF 影響癌細胞生長......................... 21
2.6 黑色素瘤各分期及對應療程........................ 21
2.7 Summary ................................. 23
第三章 材料與方法 ................................. 24
3.1 實驗設計................................. 24
3.2 實驗材料................................. 25
3.2.1 細胞培養............................... 25
3.2.2 共培養系統............................... 25
3.2.3 非熱微電漿系統........................... 26
3.3 分析儀器及方法............................. 27
3.3.1 Fiber optic thermometer......................... 27
3.3.2 光學放射性光譜(Optical emission spectrum).............. 27
3.3.3 MTS assay ............................... 27
3.3.4 Extracellular ROS 測量......................... 28
3.3.5 Intracellular ROS 測量........................ 29
3.3.6 CFSE labeling.............................. 29
3.3.7 流式細胞儀區別共培養系統中的兩細胞.............. 30
3.3.8 細胞凋亡分析............................ 31
第四章 電漿參數及產生物種分析 ........................ 33
4.1 電漿溫度................................. 33
4.2 Optical emission spectrum of He micro-plasma.............. 34
4.2.1 Wide scanning of He plasma jet ..................... 34
4.2.2 Semi-quantitation of species generated by plasma ............ 35
4.3 ROS level in the cell-containing medium in the mono- and co-culture system
under He plasma exposure ............................. 36
4.3.1 ROS level in the mono-culture system ................. 36
4.3.2 ROS level in the co-culture system .................. 38
4.3.3 不同工作氣體之ROS 濃度比較.................... 39
4.4 Summary ................................. 41
第五章 電漿對於共培養及單培養細胞之比較................... 42
5.1 電漿對單培養系統細胞生長之探討................... 42
5.2 電漿對共培養系統細胞生長之探討................... 43
5.2.1 共培養系統受電漿處理後之細胞生長曲線及細胞增生變化.... 44
5.2.2 共培養系統受電漿處理後之細胞凋亡分析............. 46
5.3 兩種培養系統對細胞影響之比較.................... 48
5.3.1 兩種培養系統不受電漿影響之細胞成長狀況............. 48
5.3.2 兩種培養系統受電漿影響後的細胞存活率比較........... 49
5.4 單次電漿處理 vs 二次電漿處理 對共培養細胞之影響........ 50
5.4.1 單次電漿處理 vs 二次電漿處理對共培養細胞處理之螢光圖... 51
5.4.2 單次電漿處理 vs 二次電漿處理對共培養細胞存活率之影響... 52
5.4.3 單次電漿處理 vs 二次電漿處理對共培養系統之細胞凋亡分析... 54
5.4.4 單次電漿處理 vs 二次電漿處理對共培養系統ROS 含量之比較. 56
5.5 Summary ................................. 58
結論..................................59
未來展望...............................60
參考文獻...............................61
附錄 1...............................68
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