||A Cell Viability Study of Non-Thermal Micro-Plasma Exposure on Malignant Melanoma Cells
||Department of Materials Science and Engineering
plasma activated medium
最近的研究已經證明，非熱微電漿（NTP）可以使癌細胞失去活性，並且對人體只有很小的副作用。 NTP是部分電離的氣體屬於物質的第四態，其是含有中性原子，電子和離子的能量粒子。以往NTP是直接照射於癌細胞或腫瘤，但可以照射的受傷面積和深度有限。因此近幾年研究了一種新的間接照射的方法，稱為Plasma Activated Medium（PAM）。 PAM方法是只有細胞培養基暴露於NTP，任何直接的NTP射線到目標細胞都排除掉，之後將處理過後的細胞培養基提供給目標細胞或是以注射的方式打到腫瘤中。PAM被證實具有與直接照射有著相同的抗癌作用，最重要的是PAM可以忽略暴露深度限制並覆蓋NTP無法實現的較大受傷區域例如腫瘤。然而PAM的抗癌細胞能力不如直接照射來的有效，但究竟是多低仍然未知，因此在本研究中，PAM的能力將通過使用直接照射作為參考與PAM比較後得到確認。本研究中使用了惡性黑色素瘤細胞B16-F10，A375和MeWo細胞，惡性黑色素瘤是最危險的皮膚癌細胞類型，選擇皮膚癌作為目標細胞的原因是由於惡性黑色素瘤可通過直接照射或PAM輕易獲得。實驗結果PAM的效果僅略低於直接照射。此外NTP對B16-F10和MeWo細胞有很大影響。另一方面A375細胞則表現出對NTP有極強的抗性。PAM是一種新的電漿方法，它有很大的淺力可以成為未來皮膚癌治癒的新選擇。
Recent researches have proven that non-thermal micro-plasma (NTP) can inactivate cancer cells with low side effects to the human body. NTP is a gas that ionized partially which contain energy particles including neutral atoms, electrons, and ions. Previously NTP was directly applied to cells or tumors but the injured area and depth is limited; hence, a new indirect plasma exposure method was investigated which known as Plasma Activated Medium (PAM). The PAM method means only medium was exposed to NTP any direct exposure from NTP to target cells was excluded; the exposed medium was then provided to target cells or injected into tumors. PAM was confirmed having the same anti-cancer effect as direct plasma exposure, most importantly PAM can ignore the exposure depth limit and cover a larger injured area that direct plasma exposure can’t achieve. However, PAM was less effective than direct plasma exposure but how exactly lower was remain unknown, in this study the ability of PAM will be affirmed by using direct plasma exposure as a reference in contrast with PAM. Malignant melanoma cells B16-F10, A375, and MeWo cells the most dangerous type of skin cancer cells was used in this study, the reason for choosing skin cancer as target cells are due to malignant melanoma is easily reachable by either direct plasma exposure or PAM. The result shows that the effect of PAM was only briefly lower than direct plasma exposure. Furthermore, NTP has a great effect on B16-F10 and MeWo cells by both direct exposure and PAM. On the other hand, A375 cells show strong resistance to NTP. PAM is a promoting methodology it has the potential to become a new option for skin cancer cure in the future.
List of Figures IX
Chapter 1 Introduction 1
1.1 Introduction 1
1.2 Motivation 3
1.3 Background 4
1.3.1 Malignant melanoma. 4
1.3.2 Malignant melanoma therapy 4
1.3.3 Non-thermal micro-plasma system 5
1.4 Objective 7
Chapter 2 Literature survey 8
2.1 Effect of NTP on murine melanoma B16 cells 8
2.1.1 Selective effect of NTP on murine melanoma B16 cells 8
2.1.2 Effect of NTP on murine melanoma B16 cells in vivo assays 11
2.2 Application of plasma device on melanoma cells 12
2.2.1 Effect of plasma device kINPen on melanoma cells 12
2.2.2 Study of dielectric barrier discharge plasma on melanoma cells by using indirect method 13
2.3 Application of Plasma Activated Medium (PAM) 13
2.3.1 PAM in vivo treatment 13
2.3.2 Role of ROS in PAM 15
2.4 Main species induced cell death 16
2.4.1 Main in situ OH radical created by NTP that induced cell death 16
2.4.2 H2O2 does not equal to NTP media 18
2.5 Plasma effect generated reactive species 19
2.5.1 Plasma effect generated reactive oxygen species (ROS) in culture medium 19
2.5.2 Intracellular ROS generated by plasma effect 20
2.6 Cell lines that show resistance to plasma exposure 21
2.7 Plasma jet kINPen based on DIN-specification 91315 22
2.8 Cell apoptosis 23
2.9 Plasma 24
2.9.1 Plasma basic principles 24
2.9.2 Plasma species and effects 25
2.10 Plasma induced ROS/RNS in cell 26
Chapter 3 Materials and methods 28
3.1 Experimental design 28
3.2 Experimental materials 29
3.2.1 Cell culture 29
3.2.2 Preparation of plasma-activated medium(PAM) 30
3.2.3 Non-thermal micro-plasma system 31
3.3 Analysis 32
3.3.1 Fiber optic thermometer 32
3.3.2 Optical emission spectrum 32
3.3.3 ROS detection 33
3.3.4 MTS assay 34
3.3.5 LDH assay 35
3.3.6 Flow cytometry 35
3.3.7 Morphology change after plasma exposure 36
3.4 Statistical analysis 36
Chapter 4 Micro-plasma diagnostic and reactive plasma species 37
4.1 Plasma temperature 37
4.1.1 Discussion for plasma temperature under DIN-specification 91315 38
4.2 Optical emission studies of non-thermal micro-plasma 38
4.2.1 Result of OES scan range: 190-1100 nm 38
4.2.2 Semi-quantitation of species generated by kINPen with He gas 39
4.2.3 Safety of UV radiation under DIN-specification 91315 40
4.2.4 Conclusion for OES scan on plasma radicals and safety of UV radiation 41
4.3 Extracellular ROS concentration change after plasma exposure 42
4.3.1 Discussion for increase in extracellular ROS after plasma exposure 44
Chapter 5 Study of NTP and PAM on malignant melanoma 45
5.1 Cell viability the MTS assay 45
5.1.1 Discussion for cell proliferation rate after plasma exposure 48
5.2 Cell cytotoxicity the LDH assay 49
5.2.1 Discussion for cell cytotoxicity rate after plasma exposure 52
5.3 Flow cytometry 53
5.3.1 B16-F10 cells show a high apoptosis rate after plasma exposure 54
5.3.2 At short plasma exposure time period MeWo cells show resistance 56
5.3.3 Amelanotic melanoma cell line A375 shows strong resistance to plasma exposure 58
5.4 Morphology change after plasma exposure in short time period 60
5.4.1 Cell become rounded after plasma exposure 61
Chapter 6 Conclusion 64
Chapter 7 Perspective 65
Chapter 8 References 66
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