저농도에 따른 사이클로스포린의 인체 각막상피세포에 미치는 영향
Effects of Low-Dose Cyclosporine on Human Corneal Epithelial Cells
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목적: 농도에 따른 사이클로스포린이 인체의 각막상피세포에 미치는 영향에 관하여 알아보고자 하였다. 대상과 방법: 인체 각막상피세포를 배양하여 1 ug/ml (0.0001%), 10 ug/ml (0.001%), 100 ug/ml (0.01%), 500 ug/ml (0.05%) 농도의사이클로스포린에 각각 5분, 10분간 접촉시킨 후 MTT 분석법으로 세포 증식 억제 정도와 LDH 분석법으로 세포의 손상 정도를 측정하였다. PIP와 Laminin으로 각막상피세포의 세포 외 기질 양을 농도별로 비교, 측정하였다. 또한, 각막상피세포에서 분비되는 TNF-α및 IL-1을 측정하여 분비되는 사이토카인의 양도 비교하고자 하였다. 결과: MTT 분석법 상 사이클로스포린의 농도와 접촉시간에 따른 유의한 차이가 없었다(p＞0.05). LDH의 수치는 대조군에 비해 모든실험군에서 유의하게 증가하였으나(p＜0.017), 노출시간에 따른 유의한 차이는 없었다(p＞0.05). 사이크롤스포린의 농도가 0.01% 이상에서는 각막상피세포에서 생성되는 세포 외 기질인 PIP나 laminin 생성에 대한 억제효과가 대조군에 비해 유의하게 나타났지만(p ＜0.028, p＜0.031), 농도가 0.01% 이하이거나 노출시간을 다르게 하여도 대조군과 유의한 차이는 없었다(p＞0.05). 염증성 사이토카인인 TNF-α와 IL-1의 각막상피세포에서의 생성억제는 농도와 노출시간에 차이가 없었다(p＞0.05). 결론: 각막상피세포에서 사이클로스포린을 사용할 경우 세포 외 기질인 PIP나 laminin 생성을 억제하거나 세포증식을 억제하기 위해서는 최소한 0.01% 농도의 노출이 필요하며, 저농도 사이클로스포린이 염증성 사이토카인 TNF-α와 IL-1에 미치는 영향력은 미비하였다.
Abstract
Purpose: To evaluate the response and cellular changes of cultured human corneal epithelium according to the concentration of topical cyclosporine. Methods: Human corneal epithelial cells were exposed to cyclosporine A concentrations of 1 ug/ml (0.0001%), 10 ug/ml (0.001%), 100 ug/ml (0.01%), and 500 ug/ml (0.05%) for 5 and 10 minutes. An MTT-based colorimetric assay was performed to assess the metabolic activity of cellular proliferation and a lactate dehydrogenase (LDH) leakage assay was used to determine cellular toxicity. The modulations of extracellular matrix proteins such as PIP and laminin were evaluated. The levels of pro-inflammatory cytokine, TNF-α, and IL-1 were evaluated by ELISA kits. Results: The inhibitory effects of human corneal epithelial cellular proliferation did not show a concentration- or exposure time-dependent response. Activity of LDH did not show a statistically significant difference for the different concentrations and exposure times. The inhibitory effects of extracellular matrix proteins such as PIP or laminin synthesized from human corneal epithelial cells were compared with those in the control group and showed a statistically significant difference at a cyclosporine concentration greater than 0.01%. Released TNF-α and IL-1 from human corneal epithelial cells did not show any difference according to concentration or cyclosporine exposure time. Conclusions: To modulate human corneal epithelial cellular proliferation and the levels of extracellular matrix proteins such as PIP and laminin, a concentration of cyclosporine A greater than 0.01% for longer than 5 minutes of exposure is needed. There were little effects of cyclosporine A on pro-inflammatory cytokine secreted from corneal epithelial cells.
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