인체 테논낭 섬유아세포에 대한 닐로티닙의 항섬유화 효과open accessThe Anti-fibrotic Effect of Nilotinib on Tenon’s Capsule Fibroblasts in Vitro
- Authors
- 강정우; 정재훈; 문남주
- Issue Date
- Jun-2018
- Publisher
- 대한안과학회
- Keywords
- Fibrosis; Glaucoma surgery; Human Tenon’s capsule fibroblast; Nilotinib
- Citation
- 대한안과학회지, v.59, no.6, pp 549 - 555
- Pages
- 7
- Journal Title
- 대한안과학회지
- Volume
- 59
- Number
- 6
- Start Page
- 549
- End Page
- 555
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/2741
- DOI
- 10.3341/jkos.2018.59.6.549
- ISSN
- 0378-6471
2092-9374
- Abstract
- 목적: 본 연구에서는 인체 테논낭 섬유아세포에 대한 닐로티닙의 항섬유화 효과를 확인하고자 하였다.대상과 방법: 녹내장수술 도중 채취하여 일차배양한 인체 테논낭 섬유아세포를 1, 5, 10, 20 μM 농도의 닐로티닙에 24시간 동안 노출시켰다. 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) assay를 이용하여 세포의 생존 정도를 측정하였고, annexin V/propidium iodide (PI) 이중염색 후 유세포분석을 시행하여 세포자멸사 정도를 확인하였다. 이후 세포자멸사 관련 단백질을 웨스턴블롯 분석 방법으로 정량적으로 평가하였다.결과: MTT assay 결과, 닐로티닙은 10, 20 μM의 농도에서 인체 태논낭 섬유아세포에 대한 항증식 효과를 보였다(p<0.001, p<0.001, 각각). Annexin V/PI 이중염색 후 시행한 유세포분석상 닐로티닙은 세포자멸을 증가시켰다. 또한 닐로티닙은 caspase-3, -9, poly adenosine diphosphate ribose polymerase의 분열을 활성화시켰으며, B-cell lymphoma-extra large, Bax 발현을 억제하였다. 이는 닐로티닙에 의해 유도되는 세포 자멸사가 미토콘드리아 경로를 통해 매개된다는 것을 의미한다. 닐로티닙은 α-smooth muscle actin, transforming growth factor beta의 발현 역시 감소시켰다.결론: 본 연구를 통해 닐로티닙은 인체 테논낭 섬유아세포의 세포 생존을 저하시키며, 미토콘드리아 경로 매개 세포자멸사를 유도하는 것을 확인하였다. 본 연구의 결과를 토대로, 녹내장수술 후 발생하는 섬유화 문제의 해결책으로서 닐로티닙이 사용될 수 있을 것이라 기대된다.
Purpose: To evaluate the anti-fibrotic effects of nilotinib on the survival of cultured human Tenon's capsule fibroblasts (HTFs).Methods: HTF primary cultures were obtained from samples following glaucoma surgery. Primarily cultured HTFs were exposed to 1, 5, 10, and 20 μM nilotinib for 24 hours. The effects of nilotinib on HTF proliferation and cell viability were determined using the 3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, and apoptosis was determined by flow cytometry using annexin-V/propidium iodide (PI) double staining. Apoptosis-related proteins were detected by western blotting.Results: The MTT assay showed that nilotinib induced an inhibition of HTF proliferation at concentrations of 10 and 20 μM (p < 0.001 and p < 0.001, respectively). Annexin V/PI double staining showed significantly increased apoptosis in cells treated with nilotinib. Nilotinib activated caspase-3, -9, and poly adenosine diphosphate ribose polymerase cleavage, and downregulated the expression of B-cell lymphoma-extra large and Bax, which indicated that nilotinib-induced apoptosis was partly mediated through the mitochondrial pathway. In addition, treatment with nilotinib decreased the expression of α-smooth muscle actin and transforming growth factor-β.Conclusions: Nilotinib decreased cell survival of cultured HTFs and induced mitochondria-mediated apoptosis. The results suggested that nilotinib may exert antiproliferative effects on HTFs, making it a possible agent to control postoperative fibrosis in patients undergoing glaucoma surgery.
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