마이토마이신, 덱사메타존, 사이클로스포린 0.05%가 인간 각막실질세포에 미치는 영향
Effect of Mitomycin C, Dexamethasone, and Cyclosporine A 0.05% on the Proliferation of Human Corneal Keratocytes
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목적: 마이토마이신C, 덱사메타존, 사이클로스포린이 인간각막실질세포에 미치는 영향을 비교하고자 하였다. 대상과 방법: 인간각막실질세포를 배양한 후 0.05% 마이토마이신C, 0.05% 덱사메타존, 0.05% 사이클로스포린에 3, 5, 10분간 접촉시켰다. MTT 분석법으로 각막실질세포의 증식억제력을 확인하였고, 콜라겐 및 Laminin 생성정도를 비교하였다. 젖산탈수소효소 분석법을 이용해 세포사 정도를 측정하였으며, 약제노출 시간에 따른 세포자멸사 유무를 확인하기 위해 유세포 분석기를 시행하였다. 결과: 세 약제 모두에서 노출시간이 증가할수록 대사활성도가 감소하고, 세포독성이 증가하는 경향을 보였다. 세포외기질 생성의 억제및 세포의 손상 정도는 마이토마이신C 및 덱사메타존이 사이클로스포린에 비해 크게 나타났다. 세포자멸사 또한 세 약제 모두 노출시간에 따라 증가됨을 알 수 있었고 사이클로스포린이 마이토마이신C, 덱사메타존보다 약하게 나타났다. 결론: 각막실질세포 증식억제는 세포자멸사와 연관성이 있으며, 각막혼탁을 줄이기 위해서는 마이토마이신C 및 덱사메타존이 사이클로스포린보다 효과적일 것으로 생각하며, 사이클로스포린의 경우 각막혼탁을 줄이기 위해서는 0.05% 이상의 고농도가 필요할 것으로생각한다.
Abstract
Purpose: To investigate the biologic effect of mitomycin C, dexamethasone and cyclosporine A 0.05% on cultured human keratocytes in vitro. Methods: Human corneal keratocytes were exposed to a concentration of mitomycin C (0.05%), dexamethasone (0.05%)and cyclosporine A (0.05%) for a period of 3, 5, and 10 minutes. MTT-based colorimetric assay was performed to assess the metabolic activity of cellular proliferation and the concentration of type I procollagen COOH-terminal peptide (PIP) and laminin were measured. Cell damage was determined by using the lactate dehydrogenase (LDH) assay. Apoptotic response was evaluated utilizing flow cytometric analysis with Annexin V and propiodium iodide. Results: The inhibitory effect of cellular proliferation and cytotoxicity in cultured human keratocytes showed a time-dependent response in all drugs. The production of PIP and laminin showed a time-dependent response in cultured cells. Apoptosis was observed in flow cytometry after being treated with mitomycin C, dexamethasone and cyclosporine A. Cyclosporin A resulted in less apoptosis of keratocytes than mitomycin C and dexamethasone. Conclusions: The apoptotic response of mitomycin C, dexamethasone and cyclosporine A is associated with the inhibitory effect of human corneal keratocyte proliferation. To decrease corneal opacity, mitomycin C and dexamethasone were more effective than cyclosporine A in the present study. Additionally, a high concentration of cyclosporine A greater than 0.05% is necessary to lower corneal opacity.
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