Yokoyama Tomoko, Yamane Ken, Minamoto Atsushi, Tsukamoto Hidetoshi, Yamashita Hidetoshi, Izumi Shunsuke, Hoppe George, Sears Jonathan E, Mishima Hiromu K
Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Exp Eye Res. 2006 Sep;83(3):602-9. doi: 10.1016/j.exer.2006.02.016. Epub 2006 May 11.
We investigated the differential protein expression patterns of retinal pigment epithelial (RPE) cells exposed to increased glucose concentrations. Cultured human RPE cells (ARPE-19) were exposed for 4 days with normal blood glucose concentration (5.5 mM D-glucose), followed by exposure to either normal (5.5 mM) or high (33 mM) concentrations of D-glucose for 48h. Protein extracts of glucose-treated RPE cells were then subjected to comparative proteome analysis based on 2-D gel electrophoresis. Protein spots were visualized by silver staining. The differentially expressed proteins were excised and digested in-gel with trypsin, then analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The expression levels of cathepsin B, glutathione peroxidase and heat shock protein 27 were increased, and that of protein disulfide isomerase decreased in high glucose treated RPE compared to normal glucose. The isoelectric point of copper/zinc-containing superoxide dismutase (Cu/Zn-SOD) shifted toward acidic region in response to high glucose. Cu/Zn-SOD activity in high glucose group was significantly lower than that in normal glucose group (P<0.05, Mann-Whitney U-test). Systematic survey of protein expression has revealed that RPE cells respond to acute, pathologically high glucose levels by the elevated expression of anti-oxidant and proteolytic enzymes.
我们研究了暴露于葡萄糖浓度升高环境下的视网膜色素上皮（RPE）细胞的差异蛋白质表达模式。将培养的人RPE细胞（ARPE - 19）先在正常血糖浓度（5.5 mM D - 葡萄糖）下培养4天，然后分别暴露于正常（5.5 mM）或高（33 mM）浓度的D - 葡萄糖中48小时。随后，对经葡萄糖处理的RPE细胞的蛋白质提取物进行基于二维凝胶电泳的比较蛋白质组分析。蛋白质斑点通过银染可视化。差异表达的蛋白质点被切下，在胶内用胰蛋白酶消化，然后通过基质辅助激光解吸/电离飞行时间质谱（MALDI - TOF MS）进行分析。与正常葡萄糖处理相比，高葡萄糖处理的RPE中组织蛋白酶B、谷胱甘肽过氧化物酶和热休克蛋白27的表达水平升高，而蛋白质二硫键异构酶的表达水平降低。含铜/锌超氧化物歧化酶（Cu/Zn - SOD）的等电点在高葡萄糖作用下向酸性区域偏移。高葡萄糖组的Cu/Zn - SOD活性显著低于正常葡萄糖组（P < 0.05，Mann - Whitney U检验）。蛋白质表达的系统研究表明，RPE细胞通过抗氧化和蛋白水解酶表达的升高来响应急性、病理性的高葡萄糖水平。
