The effects of antioxidants on pyridinoline cross linkage formation in human fibroblasts culture from hypertrophic scars

Abstract

目的: 用人皮肤瘢痕组织的成纤维细胞培养物测试抗氧化剂对吡啶连接链形成的抑制作用 ,从而筛选出有效而且不损害细胞的抗氧化剂 ,以应用于增生瘢痕的临床治疗。 方法: 用烧伤病人皮肤瘢痕组织培养成纤维细胞 ,在第 8代传代培养物中加入自由基生成系统及不同的抗氧化剂 :超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化酶、谷胱甘肽、去铁铵 ,而对照组不加任何作用物。在37℃作用 4h后 ,显微镜下观察细胞的形态变化并测定细胞及其上清液的吡啶连接链含量。 结果: 从各实验组成纤维细胞中测出的吡啶连接链的含量均低于对照组 ,尤其以过氧化氢酶、超氧化物歧化酶和去铁铵为显著 (P <0 .0 5 ) ,而其中只有在加入过氧化氢酶的细胞培养物能保持正常的细胞形态 ,其余的抗氧化剂则出现明显的细胞损害 ,表现在细胞膜破坏、细胞溶解。 结论: 过氧化氢酶等 5种抗氧化剂都能有效地抑制吡啶连接链的形成 ,在临床应用上首选过氧化氢酶 ,因其既能有效地清除自由基从而抑制吡啶连接链的形成 ,而且不损害细胞形态结构 Objective: To investigate the effects of antioxidants on the pyridinoline crosslink formation in human fibroblasts culture from hypertrophic scars. The role of these antioxidants in reducing the level of pyridinoline crosslinks in vitro without damaging the cells has important implications. These antioxidants can be applied for the treatment of hypertrophic scars. Methods: 9 cases of hyprotrophic scar biopsies were taken from burn patients. The specimens were explant culture for 7 days. Insolated fibroblasts from these explant cultures were subcultured for 8 passages. At the last passage, the cultures were subjected to a free radical generating system and were added different antioxidants. The supernatant and cells were collected after 4 hrs of incubation and the concentration of pyridinoline crosslinks was determined using Pyrilink kit. Results: When the fibroblast cultures were subjected to free radicals and antioxidants, all samples showed a decrease in pyridinoline crosslinks concentration. The more potent antioxidants found in this study were catalase, superoxide dismutase and desferrioxamine. Particularly catalase which did not damage the cells. Conclusion: The antioxidants used in this study were found to be effective in reducing the concentration of pyridinoline crosslinks. The best agent is catalase, which did not damage the cells and could be considered for clinical use. 还原