低溫氦氣常壓電漿生成之活性氧化物質分析以及電漿於生物溶液中生成氫氧自由基和過氧化氫對牙髓幹細胞培養之影響

Abstract

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.