体外培养人角膜内皮细胞的UVB损伤及抗坏血酸的抗氧化保护作用研究

以非转染人角膜内皮(HCE)细胞系为体外实验模型系统研究了UVB氧化损伤、Asc抗氧化保护及其分子机理。体外培养的HCE细胞经UVB和/或Asc处理后,利用MTT和光镜对细胞的活力和形态进行了检测,利用8-羟基脱氧鸟苷免疫荧光染色对DNA的氧化损伤进行了检测,并利用二氢乙啶染色对胞内活性氧(ROS)的水平进行了检测。结果显示,100~800 mj/cm²的UVB辐射能剂量和时间依赖性地损伤HCE细胞的活力;200 mj/cm² UVB(自然太阳光中的平均辐射剂量)能引起HCE细胞发生皱缩,并显著增加细胞的DNA氧化损伤程度及胞内ROS水平;而1 mmol/L Asc不仅能显著增强HCE细胞的活力、促进细胞分裂,而且还能显著降低200 mj/cm² UVB所引起的DNA氧化损伤及胞内ROS水平。综上所述,UVB通过诱导ROS的产生进而引起DNA氧化损伤,对HCE细胞具有显著的氧化损伤作用;而Asc能够通过降低UVB诱导的ROS水平进而保护DNA免受氧化损伤,对HCE细胞的UVB损伤具有一定的抗氧化保护作用。本文研究结果对于利用Asc等抗氧化保护剂保护HCE细胞免受UVB氧化损伤具有一定的理论指导价值。

UVB damage and protection effect of ascorbic acid on human corneal endothelial cells in vitro

To postulate the damage effect of UVB and the protection effect of antioxidant ascorbic acid(Asc), the oxidantive effect of UVB, the protective effect of Asc, and the related molecular mechanisms were investigated systematically using an in vitro model of non-transfected human corneal endothelial(HCE) cells in this study. After HCE cells were exposed to UVB irradiation and/or Asc, the viability and morphology were examined by MTT assay and light microscopy, DNA oxidative damage was detected by 8-hydroxydeoxyguanosine immunofluorescent staining, and the level of reactive oxygen species(ROS) was assayed by dihydroethidium staining. Our results showed that 100~800 mj/cm² UVB irradiation could decline the viability of HCE cells in a dose-and time-dependent manner. UVB at 200 mj/cm²(the average dose of UVB in natural sunlight) could induce cell shrinkage, oxidative damage of DNA, and elevation of ROS level significantly. Whereas, 1 mmol/L Asc, with significant enhancing effects on the viability and proliferation of HCE cells, could significantly reduce the oxidative damage of DNA and the ROS level in 200 mj/cm² UVB-irradiated HCE cells. In conclusion, UVB irradiation has a significant damaging effect on HCE cells by inducing DNA oxidative damage via ROS generation, while Asc has an obvious protecting effect on UVB-irradiated HCE cells by eleminatingDNA oxidative damage via inhibiting ROS generation. These findings can be used as certain theory guidance in protecting HCE cells from UVB-induced oxidative damage by employing Asc and the other antioxidants.