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목적: 말초혈액단핵구와 화학적으로 손상된 각막세포의 혼합반응에 대한 시스테아민의 효과를 알아보고자 하였다. 대상과 방법: 사람각막간질세포를 0.05 N NaOH로 60초간 처리하여 화학적으로 손상시킨 후 말초혈액단핵구자극 검사를 시행하였다. 혼합 말초혈액단핵구-각막세포반응은 이후 다양한 농도의 시스테아민(0-10 mM)으로 처리되었다. 세포 내 활성산소물질 형성 정도는산화에 민감한 형광탐침자인 2′,7′-dichlorofluorescein diacetate (DCF-DA)를 사용하여 측정되었다. NaOH로 처리된 각막세포에 의해 촉진된 말초혈액단핵구의 증식 속도와 염증성 사이토카인 분비는 각각 bromodeoxyuridine 증식분석검사와 효소결합 면역흡수분석법으로 측정하였다. 결과: 말초혈액단핵구 증식은 시스테아민의 농도에 비례하여 억제되었다(p=0.019). DCF-DA 형광은 시스테아민 농도에 비례하여 감소하였다(p＜0.001). 시스테아민의 농도에 비례하여 기질분해효소-9 (matrix metalloprotease-9, MMP-9), 인터류킨-6 (interleukin-6, IL-6), 형질전환성장인자-베타1 (transforming growth factor-beta 1, TGF-β1)은 억제되었고(p＜0.05), 대식세포이동저해인자(macrophage migration inhibitory factor, MIF)는 증가하였다(p=0.008). 결론: 시스테아민이 활성산소물질의 억제를 통하여 화학적으로 손상된 각막세포에 의해 유발된 말초혈액단핵구의 증식과 염증 사이토카인의 분비를 억제하였다. 따라서 시스테아민이 화학적 각막손상 환자에서 치료제로 사용될 수 있을 것으로 기대된다.
Purpose: To investigate the effect of cysteamine on mixed peripheral blood mononuclear cells (PBMCs)-chemically injured keratocytes reaction (mixed lymphocyte-keratocyte reaction; MLKR). Methods: PBMC stimulation assay was performed after keratocytes were chemically injured with 0.05 N NaOH for 60 seconds. MLKR was treated with various concentrations of cysteamine (0-10 mM). Intracellular reactive oxygen species (ROS) formation was measured using the oxidation-sensitive fluorescent probe, 2′7′-dichlorofluorescein diacetate (DCF-DA). Proliferation rate of PBMCs stimulated by NaOH-treated keratocytes and secretion profiles of matrix metalloprotease-9 (MMP-9), transforming growth factor-beta1 (TGF-β1), interleukin-6 (IL-6), and macrophage migration inhibitory factor (MIF) were determined using the bromodeoxyuridine proliferation assay and enzyme-linked immunosorbent assay, respectively. Results: Proliferation rate of PMBCs was suppressed by cysteamine in a dose-dependent manner (p = 0.019). Fluorescence of DCF-DA decreased depending on cysteamine concentration (p < 0.001). MMP-9, IL-6 and TGF-β1 levels were suppressed by cysteamine in a dose-dependent manner (p < 0.05), whereas MIF levels increased with cysteamine concentration of 0.5-10 mM (p = 0.008). Conclusions: These study results indicate that cysteamine induced the ROS-mediated inhibition of inflammatory cytokine release and proliferation of PBMCs stimulated by chemically injured keratocytes. Thus, cysteamine can be used in the treatment of chemical corneal burns.
