臺灣博碩士論文加值系統

過去有研究指出，電漿可抑制細胞再生，促進幹細胞分化，因而帶出電漿在組織再生工程應用上的可能性。除此之外，亦有研究指出，電漿所生成之活性氧化物質（Reactive Oxygen Species, ROS）會影響哺乳細胞內部的訊號傳遞網路；近期更有研究指出，ROS會激發潛在生長因子，使其轉換成生長因子，進而促使牙髓幹細胞（Dental Pulp Stem Cells, DPSCs）分化，此發現可望應用到幹細胞治療上，促使牙本質再生。而電漿中即含有前述所提之粒子，因此，本研究利用低溫常壓氦氣電漿束（Helium-based Low Temperature Atmospheric-Pressure Plasma Jet, He APPJ）進行實驗。本實驗分析電漿束中所含之ROS比例，並針對不同溶液包含去離子水（DI water）、磷酸鹽緩衝液（Phosphate Buffered Saline, PBS）以及DMEM（Dulbecco's Modified Eagle Medium），以電漿處理不同時間（10秒至60秒），量測溶液中可能造成幹細胞分化之氫氧自由基（Hydroxyl Radical, OH Radical）、過氧化氫（Hydrogen Peroxide, H2O2）之濃度，並觀察He APPJ對牙髓幹細胞在存活率，以及分化上的影響。電漿特性部分，光譜分析儀（Optical Emission Spectroscopy, OES）結果顯示在波長309 nm（氫氧自由基放光處）有明顯放光，且在電漿和溶液液面接觸面上，氫氧自由基放光強度較空氣中更為劇烈，顯示溶液中可能有更多氫氧自由基生成。而氫氧自由基以及過氧化氫量測結果皆顯示，He APPJ能夠在各溶液中生成氫氧自由基和過氧化氫，其濃度隨照射時間增加而提高，且濃度會受到溶液中成分影響而產生差異；在細胞特性部分，實驗結果指出，在一定處理時間下，He APPJ對於細胞存活率無明顯影響，且當處理時間在20至40秒時，皆可看到鹼性磷酸酶（Alkaline Phosphatase）的活性表現量明顯增加，從此結果推測He APPJ在各溶液中產生之氫氧自由基與過氧化氫極有可能為造成牙髓幹細胞分化的關鍵。

Past study have pointed out that plasma can inhibit cell proliferation, promote stem cell differentiation, which bring out the possibility of plasma on tissue regeneration engineering applications. In addition, previous study showed that the reactive oxygen species (ROS) produced by cold plasma would influence the inter- and intracellular networks on mammalian cells. Other study indicates that latent transforming growth factor-β1 can be induced by ROS to direct dental stem cells differentiation and apply to stem cell therapy to induce dentin regeneration. In this study, we propose to treat the dental pulp stem cells (DPSCs) with Helium-based low temperature atmospheric-pressure plasma jet (He APPJ) and analyze the differentiation and viability results. We also measure the concentration of hydroxyl radical (OH radical) and hydrogen peroxide (H2O2), which may be crucial factors to enhance differentiation of DPSCs , in deionized water (DI water), phosphate buffered saline (PBS) and dulbecco's modified eagle medium (DMEM) with different plasma treatment time, from 10 to 60 seconds. Optical emission spectroscopy (OES) results show that there is high intensity at 309 nm, which represents OH radical. Also, the intensity of OH radical at the interface is higher than that in free jet, shows that OH radical is enhanced at interface of gas and solution. OH radicals and H2O2 measurement results indicate that He APPJ is able to generate OH radical and H2O2 in the solution, and the concentration increases with plasma treatment time. The cell experimental results indicate that viability is not significantly changed in appropriate conditions, and the treatment of APPJ for 20 to 40 seconds enhanced differentiation of DPSCs. From the results, OH radicals and H2O2 generated by APPJ in solution may be the key points to enhance DPSCs differentiation and reveal the application potential of APPJ in dental field.

摘要 iii
ABSTRACT iv
誌謝 v
目錄 vi
圖目錄 ix
第一章、緒論 1
1.1 研究背景 1
1.1.1 齟齒治療簡介 1
1.1.2 幹細胞簡介 1
1.1.3 牙齒再生簡介 2
1.1.4 常壓電漿簡介 2
1.1.5 活性氧化物質對細胞之影響 3
1.2 文獻回顧 3
1.2.1 常壓電漿促進肺癌細胞凋亡之效果 4
1.2.2 常壓電漿束對於牙周膜間質幹細胞之影響 4
1.2.3 雷射光激發潛在生長因子誘導牙齒幹細胞分化 4
1.3 研究動機與目標 5
1.4 論文架構 5
第二章、實驗原理 7
2.1 電漿生成原理 7
2.2 光譜分析儀（Optical Emission Spectroscopy, OES） 9
2.3 傅立葉轉換紅外線光譜儀（Fourier Transform Infrared spectroscopy, FTIR） 10
2.4 相增強電荷耦合元件（Intensified Charge-coupled Device, ICCD） 11
2.5 水溶液中氫氧自由基量測 12
2.6 水溶液中過氧化氫量測 13
2.7 細胞存活率（活性）檢驗 14
2.8 細胞分化力檢驗 15
第三章、實驗方法 16
3.1 實驗流程與參數 16
3.2 細胞取得與培養 17
3.3 實驗設備 18
3.3.1 低溫常壓氦氣電漿設備 18
3.3.2 電源供應器 20
3.3.3 氣體管路系統 22
3.4 實驗分析方法與量測儀器 23
3.4.1 實驗量測儀器 23
3.4.1.1 光譜分析儀（OES） 23
3.4.1.2 傅里葉轉換紅外光譜（FTIR） 24
3.4.1.3 相增強電荷耦合元件（ICCD） 25
3.4.1.4 水溶液中氫氧自由基量測 26
3.4.1.5 水溶液中過氧化氫量測 29
3.4.2 實驗分析方法 31
3.4.2.1 細胞活性分析 31
3.4.2.2 細胞分化程度分析 32
第四章、實驗結果與討論 33
4.1 ROS分析 33
4.1.1 低溫常壓電漿束中ROS分析 33
4.1.2 電漿束與水溶液接觸面之ROS分析 35
4.1.3 DMEM化學鍵結分析 42
4.1.4 水溶液中氫氧自由基濃度量測 43
4.1.5 水溶液中過氧化氫濃度量測 47
4.1.6 通入不同氧氣流量之電漿分析 50
4.1.7 通入不同氧氣比例之過氧化氫濃度探討 52
4.2 細胞特細分析 53
4.2.1 活性分析 53
4.2.2 細胞分化程度分析 54
4.3 各實驗結果比較與討論 55
4.3.1 溶液中氫氧自由基之濃度探討 55
4.3.2 溶液中氫氧自由基及過氧化氫濃度差異之探討 57
4.3.3 不同溶液中過氧化氫之濃度探討 58
4.3.4 細胞分析與溶液中過氧化氫濃度之探討 58
第五章、結論與未來工作 60
5.1 結論 60
5.2 未來工作 60
參考文獻 62

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