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Identification and evaluation of regenerative effect on the upper airway mucosa of non-thermal atmospheric plasma treated solution

Other Title
액상형 비열대기 플라즈마(Non-thermal atmospheric plasma treated solution)의 상기도 점막 조직 재생에 대한 효과 확인 및 평가
Authors
원, 호륜
Department
대학원 의학과
Degree
Doctor (2020)
Table Of Contents
Table of Contents
Abstract ⅰ
Table of Contents ⅲ
List of Figures ⅵ
Part I
Ⅰ. Introduction 2
Ⅱ. Materials and Methods 3
A. Cell culture 3
B. Design of non-thermal plasma treated solution (NTS) 3
C. Cell cytotoxicity activity (MTT assay) 4
D. Cell proliferation activity (BrdU assay) 5
E. Scratch wound healing assay 5
F. Transwell migration assay 5
G. Western blot 6
H. Immunofluorescence assay 6
I. Quantitative real-time PCR 7
J. Gelatin zymogram assay 7
K. In vivo study 8
L. Immuno-histochemical analysis 9
M. Statistical analysis 9
Ⅲ. Results 11
A. NTS has no cytotoxicity to the BEAS-2B cells and enhances cell proliferation 11
B. NTS enhances migration of BEAS-2B cells 12
C. NTS increases cell proliferation and migration via epidermal growth factor receptor (EGFR) activities 14
D. NTS increases cell migration via the epithelial-to-mesenchymal transition (EMT) signaling 15
E. NTS enhances matrix metalloproteinases-2 (MMP-2)/matrix metalloproteinases-9 (MMP-9) activities 18
F. NTS enhances nasal mucosa wound healing in an animal model 20
Ⅳ. Discussion 23
Ⅴ. Conclusion 27
Ⅵ. References 28
Part II
Ⅰ. Introduction 34
Ⅱ. Materials and Methods 36
A. Cell culture 36
B. Fluoresce-activated cell sorting analysis 36
C. Design of non-thermal plasma treated solution (NTS) 36
D. Cell viability assay (MTT assay) 37
E. Scratch wound healing assay 37
F. Quantitative real-time PCR 37
G. Western blotting 38
H. Reverse transcription PCR 38
I. Animal model and surgical techniques 39
J. Endoscopic and histologic analysis 39
K. Immunohistochemical analysis 40
L. Videokymographic analysis 40
M. Statistical analysis 41
Ⅲ. Results 42
A. Human VF-MSCs showed multi-potency of differentiation into various tissues 42
B. NTS does not induce cytotoxicity in VF-MSCs and increases cell viability 44
C. NTS increases migration of VF-MSCs and upregulates expression of MMP-2/MMP-9 45
D. NTS reduces fibrosis-related molecules and inhibits transformation of VF-MSCs into myofibroblasts 47
E. NTS modulates ECM deposition of VF mucosa and promotes recovery of VF function in vivo 49
Ⅳ. Discussion 55
Ⅴ. Conclusion 58
Ⅵ. References 59
국문요약 63
Abstract
Plasma is a mixture of electrons, ions, and energetic photons in the form of ionized gas that is generated when the gas is further energized. Non-thermal atmospheric plasma has several medical effects such as sterilization, anti-cancer, anti-inflammation, and tissue regeneration. However, the existing probe type is limited to local direct treatment. Therefore, we investigated the various effects using liquid type plasma to overcome this limitation. In addition, the therapeutic effects of non-thermal plasma treated solution (NTS) on upper airway mucosa have yet to be determined. In the first part, the effect of NTS on the regeneration of nasal mucosa was studied. Experiments were carried out using BEAS-2B, a human bronchial epithelial cell line similar to nasal mucosa epithelium. NTS had no cytotoxicity to the BEAS-2B cells and enhanced cell proliferation. NTS also promoted migration of BEAS-2B cells. NTS increased cell proliferation and migration via epidermal growth factor receptor activities and epithelial-to-mesenchymal transition signaling. Furthermore, NTS enhanced wound healing of nasal mucosa in an animal model. In the second part, the effect of NTS on the regeneration of vocal fold mucosa was examined. Migration and matrix metalloproteinase-2 expression of lipopolysaccharide (LPS)-treated human vocal fold-derived mesenchymal stem cells (VF-MSCs) were enhanced by NTS treatment. NTS treatment not only ameliorated nuclear factor-κB and interleukin-6 activation, induced by NTS treatment, but also the increased manifestation of α-smooth muscle actin and fibronectin, induced by transforming growth factor-ß. In a rabbit vocal fold scarring animal model, histological analyses showed increased hyaluronic acid deposition and decreased collagen accumulation after NTS injection. Videokymographic analysis showed more improved vibrations in NTS-treated vocal fold mucosa compared to those in non-treated group. The results of this study suggest that NTS may improve regeneration of the upper airway mucosa and improve functional remodeling following damage to the upper airway mucosa.

플라즈마(Plasma)는 전자(Electrons)와 이온(Ions), 그리고 광자(Photons)가 이온화된 기체의 형태로 혼합한 것으로, 기체에 에너지를 추가적으로 공급하여 생성된다. 비열 대기성 플라즈마(Non-thermal atmospheric plasma : NTP)는 기존 연구를 통해서 살균, 항암, 항염증, 조직 재생 등 여러가지 의학적 효과가 있는 것으로 알려져 있다. 단, 기존의 기체 형태의 프로브 타입은 국소적인 치료를 위한 적용에는 제한점이 있다. 따라서 이러한 한계를 극복하기 위해 액체형 플라즈마(Non-thermal plasma treated solution : NTS)를 고안하였으며, 이에 상기도 점막에 대한 조직재생 효과를 확인하고자 하였다. 1부에서는 NTS가 코 점막 재생에 미치는 영향을 연구하였다. 실험은 비강점막상피와 유사한 인간 기관지 상피세포주인 BEAS-2B를 이용하여 수행하였다. NTS는 BEAS-2B 세포에 대하여 세포독성(Cell cytotoxicity)이 없었고, 세포 증식(Cell proliferation)이 촉진되었다. NTS는 또한 BEAS-2B 세포의 이주(Migration)을 촉진했다. NTS는 표피 성장 인자 수용체(Epidermal growth factor receptor) 활성과 세포 표현형 전이(Epithelial-to-mesenchymal transition) 신호를 통해 세포 증식과 이주를 증가시켰다. 게다가, NTS는 동물모델에서 코 점막의 상처치유를 개선시켰다. 2부에서는 NTS가 성대 점막의 재생에 미치는 영향을 대한 연구를 수행했다. NTS는 리포폴리사카리이드(Lipopolysaccharide : LPS) 를 처리한 인간 성대 유래 중간엽 줄기세포(VF-MSC)의 이주를 증가시켰으며, Matrix metalloproteinase-2(MMP2)의 발현이 증가하였다. NTS 치료는 Nuclear factor-κB(NF-κB)와 인터루킨-6(Interleukin-6 : IL-6)의 활성화뿐만 아니라 Transforming growth factor-ß(TGF-ß)처리에 의해 유발된 α-smooth muscle actin(α-SMA)와 fibronectin의 발현도 감소시켰다. 토끼 성대 손상 동물 모델에서 NTS 처리 후 히알루론산(Hyaluronic acid : HA)의 침전량이 증가하고, 콜라겐(Collagen)의 축적이 감소하는 것으로 나타났다. Videokymography 분석에서는 NTS 처리 후 성대 점막의 진동이 대조군의 진동에 비해 더 향상되었음을 보여주었다. 본 연구 결과는 NTS가 상부 기도 점막의 재생을 개선하고 상부 기도 점막의 손상에 따른 기능적 리모델링(Remodelling)을 개선할 수 있음을 시사한다.
Keywords
Non-thermal atmospheric plasma treated solution (NTS)RegenerationNasal mucosaVocal fold mucosaWound healing액체형 비열 대기성 플라즈마(Non-thermal atmospheric plasma treat solution)재생(Regeneration)코 점막(Nasal mucosa)성대 점막(Vocal fold mucosa)상처 치유(Wound healing)
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원, 호륜
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