섬유성 망막 질환에서 전이성장인자 신호전달과정 억제를 통한 항섬유화 약물 (pirfenidone)의 효과 및 기전
Effect of Anti-fibrotic Drug (Pirfenidone) on TGF-β Signaling Pathway in Ocular Fibrotic Disorders
- 주제(키워드) Transforming growth factor-β (TGF-β) , retinal pigment epithelial (RPE) cells , fibrosis , pirfenidone , cell singnal , epithelial-mesenchymal transition
- 발행기관 아주대학교
- 지도교수 안재홍
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000020173
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Purpose: Transforming growth factor-β (TGF-β) plays a key role in transforming retinal pigment epithelial (RPE) cells into mesenchymal fibroblastic cells, which are implicated in fibrotic disorders of the retina. Herein, the effect of pirfenidone, a novel anti-fibrotic agent, on TGF-β1-mediated fibrogenesis in the human RPE cell line (ARPE)-19 was tested. Methods: The effect of pirfenidone on the TGF-β1-induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. Fibronectin and collagen production was measured with enzyme-linked immunosorbent assay, and cell migration activity was investigated using a scratch assay. Immunoblot analyses of cofilin, Smad protein (Smad) 2/3, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and extracellular signal related kinase expression were conducted to elucidate the cell signaling networks that contribute to the anti-fibrotic effect of pirfenidone. Results: Treatment with TGF-β1 induced typical phenotypic changes such as formation of stress fiber running parallel to the long axis of cells, and enhanced migration and production of extracellular matrix components such as collagen type I and fibronectin. This fibroblast-like phenotype induced by TGF-β1 was significantly inhibited by pretreatment with pirfenidone in a dose-dependent manner. Pirfenidone inhibited TGF-β signaling by preventing nuclear accumulation of active Smad2/3 complexes rather than phosphorylation of Smad2/3. Conclusions: These results collectively provide a rational background for future evaluation of pirfenidone as a potential anti-fibrotic agent for treating proliferative vitreoretinopathy and other fibrotic retinal disorders.
more목차
I. INTRODUCTION 1
A. Fibrotic Retinal Diseases 1
B. Retinal Pigment Epithelial Cells 2
C. Epithelia-to- Mesenchymal Transition 3
D. Transforming Growth Factor 4
1. Smad-Dependent Signaling 4
2. Smad-Independent Signaling 5
E. Novel Anti-fibrotic Drug (Pirfenidone) 5
F. Aims of This Study 8
II. MATERIALS AND METHODS 9
A. MATERIALS 9
1.Reagents 9
B. METHODS 9
1. Cell Cuture 9
2. Enzyme-linked Immunosorbent Assay 9
3. Immunocytochemistry 10
4. Cell Migration Assay 10
5. Immunoblot analysis 10
6. Statistical analysis 11
III. RESULTS 12
A. Pirfenidone inhibits TGF-β1 induced fibroblastic phenotypes in ARPE-19 cells 12
B. Pirfenidone suppresses the TGF-β1-induced expression of ECM components in ARPE-19 cells 22
C. Pirfenidone abrogates the TFG-β1-induced migration of ARPE-19 cells 22
D. Pirfenidone blocks TGF-β1-induced nuclear translocation but not phosphorylation of Smads 27
IV. DISCUSSION 33
A. Pirfenidone and TGF-β1 induced fibroblastic phenotypes in ARPE-19 cells 33
B. Pirfenidone and TGF-β1-induced expression of extracellular matrix components in ARPE-19 cells 35
C. Possible molecular mechanisms of pirfenidone for the inhibitory action for TGF-β1-induced fibrogenesis in ARPE-19 cells 36
V. CONCLUSION 41
REFERENCES 42
국문요약 50
