익상편조직의 섬유모세포에 미치는 사이클로스포린의 영향
Effects of Cyclosporin on Pterygium Fibroblasts
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목적: 익상편 조직의 재발 억제를 목적으로 임상에서 이용되는 사이클로스포린이 익상편조직의 세포에 미치는 영향에 관하여 알아보고자 하였다. 대상과 방법: 수술적 방법으로 절제된 인체 익상편세포를 배양하여 0.1 μg/ml (0.00001%), 1 μg/ml (0.0001%), 10 μg/ml (0.001%), 100 μg/ml (0.01%), 500 μg/ml (0.05%) 농도의 사이클로스포린에 각각 5분, 10분간 접촉시킨 후 MTT 분석법으로 세포 증식 억제 정도와 LDH 분석법으로 세포의 손상정도를 측정하였다. 익상편 세포의 세포 외 기질인 Procollagen type I COOH-terminal peptide (PIP)와 Laminin, Matrix metalloproteinase (MMP)-1, MMP-2, MMP-9의 양을 농도별로 비교, 측정하였다. 또한 익상편세포에서 분비되는 Tumor necrotic factor (TNF)-α 및 Interleukin (IL)-1b, IL-6, IL-8을 측정하여 분비되는 사이토카인의 양도 비교하고자 하였다. 결과: MTT 분석법상 대조군에 비해 농도와 노출시간에 따른 유의한 증식억제는 관찰되지 않았다(p＞0.05). LDH의 수치는 유의한 차이가 없었으며, 세포 외 기질인 PIP나 laminin, MMP-1, MMP-2, MMP-9 생성억제에 대한 차이도 없었다(p＞0.05). 전염증성 사이토카인인TNF-ɑ와 IL-1b, IL-6, IL-8의 생성억제도 대조군에 비해 농도와 약제의 노출시간에 따라 의미 있는 차이는 없었다(p＞0.05). 결론: in vitro 상태의 익상편 세포에 대해 농도 0.05% 이하, 노출시간 10분 이하의 사이클로스포린은 세포증식을 억제하거나 세포손상을 심하게 유발하지 않았으며, 세포 외 기질인 PIP나 laminin, MMPs 및 전염증성 사이토카인인 TNF-α, ILs에 미치는 영향력이 미비하였다.
Abstract
Purpose: To evaluate the response and cellular damage of cultured human pterygial cells according to the concentration and exposure time of topical cyclosporin. Methods: Human pterygial cells were exposed to a cyclosporin A concentrations of 0.1 μg/ml (0.0001%), 1 μg/ml (0.0001%), 10 μg/ml (0.001%), 100 μg/ml (0.01%), or 500 μg/ml (0.05%) for 5 or 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 damage. The extra-cellular matrix of PIP, laminin and MMP were evaluated, and the measurement of pro-inflammatory cytokine, TNF-α and IL-1b. IL-6, IL-8 was performed using ELISA kits. Results: The pterygial cellular inhibitory effect of cyclosporin was similar to that of the control according to the concentration and exposure time (p>0.05). Compared with the control, the level of LDH did not show a statistically significant difference between concentration and exposure time (p>0.05). There was no significant difference of inhibitory effects by PIP, laminin, or MMP between the experimental and control groups (p>0.05). The production of TNF-α and IL from the experimental pterygial cells due to the effect of cyclosporin was not significantly different from that of the control at a longer exposure time or stronger concentration (p>0.05). Conclusions: The response of pterygial cells to topical cyclosporin A at concentrations less than 0.05% for less than 10 minutes of exposure time showed no prevention of pterygial recurrence. With regard to cellular damage, little effects on inhibition of PIP, laminin, MMP, IL, and TNF-α were observed compared with that of the control.
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