광역학 치료에 의한 광회춘 기전에 있어 인체 진피 섬유아세포내 신호 전달 체계에 관한 연구
Yong Hyun Jang
- 주제(키워드) Photodynamic therapy , Photorejuvenation , Reactive oxygen species , Intracellular signal transduction
- 발행기관 아주대학교
- 지도교수 김유찬
- 발행년도 2011
- 학위수여년월 2011. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011766
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The clinical studies have reported over years that the photodynamic therapy (PDT) has effects on the rejuvenation of photoaged skin. However, there have been no studies on the intracellular mechanism of photorejuvenation following PDT. The purpose of this study was to investigate the effects of PDT on the signal transduction mechanism in human dermal fibroblast and the role of reactive oxygen species (ROS) in vitro. Normal human fibroblasts were treated with various sublethal and relatively low doses of 5-aminolevulinic acid (ALA) and red light. The inhibition of fibroblast proliferation was not observed at the ALA concentration of 0.1mM, ALA incubation time of 30 minutes, and 3 J/cm2 of red light energy. In this condition, I found that a small quantity of ROS that continues to be produced after 8 hours of PDT was processed with 2,7-dichlororofluorescein diacetate (DCFDA) and visualized with fluorescence microscopy and confocal microscopy. Moreover, extracellular-signal-regulated kinase (ERK) signal, among the mitogen-activated protein (MAP) kinases which is known to be associated with cell proliferation, differentiation and survival, also remained to be activated after 8 hours of PDT in the same PDT condition, like ROS, and this was confirmed through western blotting and immunohistochemical stains. The expression of transforming growth factor (TGF)-β mRNA by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was increased at 6 hours of PDT compared to that in other treatments. Matrix metalloproteinase (MMP)-1 and MMP-3 mRNA by real-time PCR significantly increased in PDT group compared with control group showing a maximal increase 24 h following PDT. TGF-β mRNA increased in PDT group at 6, 12 h post-PDT. Collagen Iα1 mRNA levels also increased after PDT in a time-dependent manner. MMP-3 and collagen Iα1 protein levels increased after PDT at 24, 48 h compared with control group. Results of this study indicate that a small quantity of ROS after PDT in dermal fibroblasts may be a critical aspect of the prolonged activation of ERK signaling that may lead to fibroblast proliferation and activation. This study provides valuable evidence of the photorejuvenation effect of PDT in vivo.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 5
A. MATERIALS 5
1. Cell culture 5
2. Fibroblast groups 5
B. METHODS 5
1. Treatment of ALA and irradiation procedure 5
2. The effects of the various PDT conditions on the
fibroblast survival 6
3. The determination of the optimal low dose-PDT
condition which does not decrease fibroblast
cellularity 6
4. Measurement of intracellular ROS by DCFDA
assay 7
5. MAP kinase assays by western blot analysis 7
6. Immunohistochemical staining of phospho-ERK 8
7. Semi-quantitative RT-PCR for TGF-β 8
8. Real-time PCR for MMP-1, 3, TGF-β, and
collagen type Iα1 9
9. Western blot analysis for MMP-1, 3, TGF-β, and
collagen type Iα1 10
10. Statistics 11
III. RESULTS 12
A. The effects of various PDT conditions on the
fibroblast survival 12
B. The determination of the optimal low dose-PDT
condition which does not decrease fibroblast
cellularity 14
C. ROS production in the non-cytotoxic, low-dose PDT
condition 16
D. Effect of low-dose PDT in non-cytotoxic condition on
MAP kinase activation 19
E. Time dependent effects of PDT on TGF-β mRNA
levels by semi-quantitative RT-PCR 21
F. Time dependent effects of PDT on the MMP 1, 3,
TGF-β, and Collagen type Iα1 mRNA levels by
real-time PCR 22
G. Time dependent effects of PDT on the MMP 1, 3,
TGF-β, and Collagen type Iα1 protein levels by
western blot analysis 24
IV. DISCUSSION 27
V. CONCLUSION 35
REFERENCES 36
국문요약 46
