长波紫外线照射对培养人表皮黑素细胞与真皮成纤维细胞抗氧化活性影响

Comparison on Catalase Protein Expression and UVA-induced DNA Damage in Cultured Human Melanocytes and Fibroblasts

目的对比观察了长波紫外线(UVA)照射对培养人表皮黑素细胞与真皮成纤维细胞过氧化氢酶(CAT)蛋白表达以及DNA氧化产物8-羟基-脱氧鸟苷(8-oxo-dG)生成的影响,旨在认识除生成黑素以外黑素细胞具有的抗氧化机制。方法建立人成纤维细胞及人黑素细胞原代和传代培养。培养细胞给予不同剂量(0,3,5,25 J/cm2)UVA照射,孵育3 h后,用间接免疫荧光的方法检测CAT和8-oxo-dG的水平。结果 3 J/cm2UVA照射后,成纤维细胞CAT蛋白表达与假照射组对比明显增加(P<0.001),8-oxo-dG荧光强度差别无统计学意义(P=0.918);黑素细胞中CAT蛋白表达与假照射组对比明显减少(P<0.001),8-oxo-dG荧光强度显著增加(P=0.002)。结论表皮黑素细胞对氧化应激存在固有的敏感性,且暴露UVA介导的氧化应激后黑素细胞缺乏适应性上调CAT表达,这些机制可能关系到白癜风皮损黑素细胞氧化损伤的发生。

Objective To investigate the changes of catalase expression and oxidative DNA product 8-hydroxy- deoxyguanosine （8-oxo-dG） in cultured melanocytes and fibroblasts in the presence or absence of UVA-mediated oxidative stress, further understanding the antioxidative activities of melanocytes except for melanin production. Methods Establish- ment of primary fibroblast cultures from adult foreskin tissue was achieved in this study. Primary melanocyte （HEMC-LP） was purchased from Cascade Co, USA and subcultured in complete M254 medium. The cells grown in cover slides were irradiated by different doses of UVA （0,3,5,25 J/cm2）. The levels of 8-oxo-dG and catalase protein in both treated and untreated cells were detected by an indirect immunofluorescence technique. Results The level of catalase protein was increased in the fibroblasts exposed to 3 J/cm2 UVA radiation in comparison with the sham-irradiated control （P〈0.001）, no difference of 8-oxo- dG immunostaining was found between irradiated and sham-irradiated group （P=0.918）. Whereas, the level of catalase protein was significantly decreased in the melanocytes exposed to 3 J/cm2 UVA radiation comparing with the sham-irradiated control （P〈0.001）, UVA-irradiated melanocytes exhibited more 8-oxo-dG immunostaining than did sham-irradiated cells （P=0.002）. Conclusion Human melanocytes are more susceptible to exogenous oxidative stress than fibroblast, adaptive upregulation of catalase is absent in melanocytes under UVA-mediated oxidative stress, which contributes to oxidative damage of melanocytes seen in vitiligo.