摘要
探讨机械牵拉对人角膜成纤维细胞氧化应激水平、抗氧化能力、炎性因子及基质金属蛋白酶(MMP)表达的影响。对体外培养的正常人(NC)及圆锥角膜患者(KC)角膜成纤维细胞进行周期性机械牵拉(幅度15%,频率0.1 Hz,时间12 h).采用荧光染色法检测活性氧自由基(ROS)水平;采用实时荧光定量PCR检测抗氧化酶基因表达;采用酶联免疫吸附试验方法检测炎性因子和MMP-9表达。结果发现:静态培养条件下, KC细胞ROS水平、白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)、MMP-9均明显高于NC细胞;与静态培养组比较,机械牵拉显著增加了NC和KC细胞ROS、炎性因子和MMP-9蛋白表达水平;并使NC细胞部分抗氧化酶基因表达上调,但不同KC患者细胞牵拉后抗氧化酶表达不同。结果提示力学刺激可能通过处于较高氧化应激状态的圆锥角膜成纤维细胞释放更多的炎性因子,进而上调MMPs的表达,加速角膜细胞外基质降解,促进圆锥角膜的发生与发展。
In order to investigate the effects of mechanical stretch on the oxidative stress, expression of inflammatory cytokines and matrix mentalloproteinase (MMP) in normal human and keratoconous patients, human normal corneal (NC) fibroblasts and keratoconic corneal (KC) fibroblasts were cultured and subjected to cyclic stretch (15% elongation, 0.1 Hz, 12 h) in vitro. The reactive oxygen species (ROS) production was measured using fluorescent dye. The expression of gene or protein of antioxidant enzymes, inflammatory cytokines, and MMP-9 was determined by real-time fluorescent quantitative PCR or enzyme-linked immunosorbent assay. Our results show that ROS level, the expression of interleukin-1β(IL-1β), IL-6, tumor necrosis factor α(TNF-α), MMP-9 in KC fibroblasts were significantly higher than those in NC ones under the static cultured condition. Compared with the static controls, cyclic stretch increased the ROS level and gene or protein expression of inflammatory cytokines in both NC and KC fibroblasts. Antioxidant enzymes were up-regulated in NC fibroblasts, but behaved differently in different KC fibroblasts after cyclic stretch. This finding suggests that mechanical stretch may up-regulate the expression of inflammatory cytokines in KC fibroblasts that are in the higher oxidative stress state. This may lead to the expression of MMPs, accelerate the degradation of extracellular matrix of the corneal tissue, and facilitate the development of keratoconus.
作者
李晓娜
宋一菲
史佩佩
宋婕
陈维毅
贺瑞
LI Xiaona;SONG Yifei;SHI Peipei;SONG Jie;CHEN Weiyi;HE Rui(College of Biomedical Engineering, Taiyuan University of Technology, Taiyuan 030024,China;Department of Excimer Laser, Shanxi Eye Hospital, Taiyuan 030002,China)
出处
《太原理工大学学报》
CAS
北大核心
2019年第4期529-535,共7页
Journal of Taiyuan University of Technology
基金
国家自然科学基金资助项目(11872262,11572213,11632013)
山西省国际合作项目(201703D421019)
关键词
角膜成纤维细胞
机械牵拉
氧化应激
炎性因子
基质金属蛋白酶
corneal fibroblasts
mechanical stretch
oxidative stress
inflammatory cytokines
matrix metalloproteinase
