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研究生:張翰倫
研究生(外文):Han-Lun Chang
論文名稱:低溫噴射式大氣電漿對纖維母細胞影響之研究
論文名稱(外文):Interaction of Non-Thermal Atmospheric-Pressure Plasma-Jet on Fibroblasts
指導教授:王明誠王明誠引用關係
指導教授(外文):Ming-Chen Wang
學位類別:碩士
校院名稱:中原大學
系所名稱:生物醫學工程研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:94
中文關鍵詞:DBD 大氣電漿纖維母細胞傷口癒合低溫噴射式電漿
外文關鍵詞:Wound HealingFibroblastsNon-thermal plasma-jetDielectric Barrier Discharge (DBD)
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低溫噴射式大氣電漿被廣泛的應用在醫療上,包含滅菌、傷口癒合以及使惡性腫瘤凋亡等。電漿被證明可以在組織上進行滅菌,但不會使組織發炎,在慢性傷口的治療上,電漿已經成為一種很有吸引力的工具。不同的電漿參數對細胞造成的影響也不同,如造成細胞增生、凋亡或是細胞膜的損傷,低劑量的電漿對血管內皮細胞是相對無毒性,而高劑量的電漿會造成內皮細胞死亡。適當的電漿參數可能會促使纖維母細胞增生,纖維母細胞的增生在傷口癒合的皮膚生物學上產生關鍵作用。本研究評估低溫噴射式大氣電漿對纖維母細胞的細胞活性、膠原蛋白分泌量、移動能力、細胞型態及巨噬細胞發炎反應,我們的目標是優化電漿處理的實驗參數,例如處理時間及外加的功率等,進而促使纖維母細胞增生,治療傷口癒合。本研究以自行設計之高壓阻尼波電源供應系統與介電質阻擋放電型式的低溫噴射式大氣電漿反應器結合,進行與細胞交互作用之研究,該設備在12.2 kV 的工作電壓與167.7 kHz的工作頻率下激發電漿,此激發狀態的電流為0.32 A,消耗功率為17.74 W,激發過程中最高溫度為25.43 oC。利用光學放射光譜儀檢測氦氣電漿可於777.32、845.74 和927.28 nm 可以觀察到活性氧物種的光譜。低劑量的電漿(90秒以下)對纖維母細胞是相對低的毒性,而高劑量的電漿(120至180秒)會造成細胞活性、膠原蛋白分泌量及移動能力下降。電漿處理10秒會促使纖維母細胞比控制組多增殖16 %的細胞活性。最後,我們成功利用適當的電漿參數電漿促使纖維母細胞增加細胞活性及移動能力。


Non-thermal atmospheric-pressure dielectric barrier discharge (DBD) plasma-jet is being widely developed for medical applications, including wound healing, malignant cell apoptosis and bacterial sterilization. The plasma process is demonstrated to cause bacterial inactivation and non-inflammatory tissue modification. It has become an attractive tool for improvement in chronic wound management. The parameters of plasma treatment influence the effect of cell, including proliferation, apoptosis and cell membrane damage. Low energy plasma is relatively non-toxic to fibroblasts while treatment at longer exposures led to cell death. Appropriate parameters of treatment will enhance cell proliferation. Proliferation of fibroblasts is a key role about wound healing in skin biology. In this study, we investigate the cell viability, collagen secretion, migration ability, cell morphology and NO release of non-thermal atmospheric-pressure helium DBD plasma-jet treatment on fibroblasts and macrophage. Our goal is to optimize plasma treatment by varying experimental parameters, such as treatment time and applied power, to enhance proliferation, collagen secretion and migration ability of fibroblasts for chronic wound healing. This study is using the damping-like factor generator to coupling high voltage coil, and combines the cold atmospheric pressure plasma-jet device for sterilization. The applied voltage and the discharge current of a damping waveform is respective 12.2 kV and 0.32 A, the frequency is 167.7 kHz, the total power of plasma-jet is 17.74 W, the maximum plasma applied temperature is respective 25.43 oC. Diagnose the active species in the helium plasma by optical emission spectroscopy (OES). The spectrum of reactive oxygen species can be observed at 777.32, 845.74 and 927.28 nm (O, OH). Low dose plasma (up to 90 s) was relatively non-toxic to fibroblast cells while treatment at longer exposures (120 s to 240 s) led to cell viability, collagen secretion and migration ability decrease. Fibroblast cells treated with plasma for 10 s demonstrated 16 % as much proliferation as untreated cells five days after plasma treatment. Finally, we get the optimization of plasma parameters for cell proliferation and migration ability on fibroblasts.


目錄

摘要 I
Abstract II
謝誌 IV
目錄 VI
圖目錄 IX
表目錄 XI
第一章 緒論 1
1-1 研究背景 1
1-2 文獻回顧 2
1-2-1低溫電漿的應用 2
1-2-2電漿傷口滅菌 2
1-2-3電漿與細胞交互作用 3
1-3 研究目的 4
第二章 理論基礎 5
2-1 傷口癒合過程 5
2-1-1 發炎時期 5
2-1-2 表皮再生 7
2-1-3 肉芽組織的形成 8
2-1-4 新生血管 9
2-1-5 傷口收縮和細胞外間質重建 10
2-2 電漿基本原理 12
2-2-1 電漿簡介 12
2-2-2 非平衡電漿 13
2-2-3 噴射式大氣壓力電漿 16
2-3 電漿檢測 17
2-3-1 Q-V Lissajous Figure 17
2-3-2光學放射光譜儀 20
2-4 電漿滅菌原理 21
2-5 電漿誘導細胞增生原理 22
第三章 材料與方法 23
3-1 研究架構 23
3-2電漿系統設計與製作 25
3-2-1 電源供應器製作 26
3-2-2 反應器設計與製作 27
3-3 電漿激發特性分析 28
3-3-1電漿電性檢測 28
3-3-4 電漿激發溫度檢測 30
3-3-5電漿物種檢測 31
3-3-6 培養基pH值評估 31
3-4細胞培養 32
3-4-1 細胞來源 32
3-4-2細胞培養藥品配置 33
3-4-3 細胞培養方法 34
3-5 電漿與細胞交互作用 37
3-5-1電漿處理細胞實驗設計 38
3-5-2 細胞活性分析 39
3-5-3膠原蛋白分泌量分析 42
3-5-4體外傷口癒合分析 43
3-5-5細胞型態分析 43
3-5-6巨噬細胞發炎反應分析 44
3-6 統計分析 44
第四章 結果與討論 45
4-1 電漿系統檢測 45
4-1-1 電漿電性檢測 46
4-1-2電漿消耗功率檢測 49
4-1-3電漿激發溫度檢測 50
4-1-4 電漿物種檢測 52
4-1-5 培養基pH值檢測 54
4-2電漿與細胞交互作用 55
4-2-1 細胞活性分析 56
4-2-2膠原蛋白分泌量分析 63
4-2-3體外傷口癒合分析 68
4-2-4 細胞型態分析 70
4-2-5 巨噬細胞發炎反應分析 72
第五章 結論與未來展望 73
5-1結論 73
5-1-1安全劑量評估 73
5-1-2體外傷口癒合表徵 73
5-2未來展望 74
參考文獻 75


圖目錄

圖2- 1 傷口癒合的發炎時期各種細胞分泌生長因子與細胞作用情形 6
圖3- 1 低溫噴射式大氣電漿對纖維母細胞影響之研究架構圖 24
圖3- 2 低溫噴射式大氣電漿系統示意圖 25
圖3- 3 電源系統方塊圖 26
圖3- 4 反應器之剖面圖 28
圖3- 5 電漿功率檢測示意圖 30
圖3- 6 血球計數盤(hemacytometer)之計數腔室示意圖 37
圖3- 7 電漿處理細胞實驗流程圖 38
圖4- 1 低溫噴射式大氣電漿激發態 46
圖4- 2 一次高壓damping波形之頻率 47
圖4- 3 群聚高壓damping波形之頻率 48
圖4- 4 每組群聚高壓damping波形出現之頻率 48
圖4- 5 電漿激發時電壓電流圖 49
圖4- 6電漿消耗功率 50
圖4- 7 電漿激發時間與電漿激發溫度之變化 51
圖4- 8 低溫噴射式大氣電漿激發態 52
圖4- 9 低溫噴射式大氣電漿激發之光譜 (200-500 nm) 53
圖4- 10 低溫噴射式大氣電漿激發之光譜 (500-1000 nm) 53
圖4- 11電漿處理時間對培養基pH值影響。 55
圖4- 12 電漿處理長時間對細胞活性影響 57
圖4- 13 電漿處理短時間對細胞活性影響 57
圖4- 14 電漿造成細胞活性下降的時間 58
圖4- 15 電漿處理時間對於纖維母細胞的細胞活性之影響 60
圖4- 16 液體體積對於細胞活性的影響 61
圖4- 17 電漿功率對於細胞活性的影響 62
圖4- 18 電漿處理30秒後培養1、3、5天之細胞存活率原始吸光值 63
圖4- 19 電漿對纖維母細胞分泌膠原蛋白能力之影響 66
圖4- 20 電漿對纖維母細胞長時間分泌膠原蛋白能力之影響 66
圖4- 21 電漿對每一纖維母細胞分泌膠原蛋白能力之影響 67
圖4- 22 電漿處理後,0至24小時細胞的移動情形 69
圖4- 23 電漿處理短時間對細胞移動能力之影響 69
圖4- 24 電漿處理長時間對細胞移動能力之影響 70
圖4- 25 電漿對纖維母細胞型態之影響 71
圖4- 26 電漿對巨噬細胞發炎反應之影響 72


表目錄

表1- 1 全球先進創傷照護技術市場 2
表2- 1 一般冷電漿的粒子溫度 14
表4- 1 電漿激發時間與電漿激發溫度之關係 51

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