过氧化氢诱导的氧化应激对ARPE-19细胞中泛素化途径的激活

Effects of hydrogen peroxide on ubquitin and ubiquitinated proteins in ARPE-19

目的探讨H2O2诱导的氧化应激对人视网膜色素上皮(RPE)细胞系ARPE-19细胞中泛素及泛素化蛋白表达的影响。方法MTT法建立H2O2诱导ARPE-19细胞模型。不同浓度H2O2作用于ARPE-19细胞后,RT-PCR检测细胞内泛素基因转录水平,细胞免疫荧光方法检测细胞内泛素化蛋白的表达。用有(或无)蛋白酶体抑制剂MG-132预处理后再用H2O2处理ARPE-19,Western blot方法检测细胞内泛素化蛋白。结果氧化应激后UBA、UBB的转录水平明显增加(P<0.05),UBC无明显变化。细胞内相对分子质量为50000～250000的泛素化蛋白均增加,MG-132预处理后再用H2O2处理,细胞内增加的泛素化蛋白则多集中在相对分子质量75000以上。细胞内泛素化蛋白表达增加,核内增加明显。结论H2O2诱导的ARPE-19氧化应激过程激活细胞内泛素化途径。

Objective Oxidative stress in retinal pigment epithelial（RPE） cells is thought to be one of the risk factors in retinal diseases because of the specific anatomic and physiological characteristics of RPE cells. The purpose of this study was to investigate the change of ubiquitination induced by oxidative stress in ARPE-19 cells. Methods ARPE-19 ceils were cultured and treated with different concentrations of hydrogen peroxide for 4 hours respectively,and the cell viability was detected by using MTT assay. The change of ubiquitin mRNA was detected by RT-PCR. MG-132, a proteasome inhibitor, was used to pretreat ARPE- 19 cells and then hydrogen peroxide was used to treat the pretreated ARPE-19 cells. The expression of ubiquitinated proteins in pretreated cells was detected by Western blotting. Intracellular distribution of ubiquitinated proteins in ARPE-19 cells treated with hydrogen peroxide was detected by immunofluorescence stain assay. Results ARPE-19 cell viability was significantly reduced after treated by 50-300 μmol/L hydrogen peroxide. The mRNA levels of ubiquitin A52 and ubiquitin B in ARPE-19 cells were obviously increased after treated with 50,100,200,300 μmol/L of hydrogen peroxide in comparison with control group （P 〈 0.05 ） ,but no statistically significant difference was seen in ubiquitin C expression（ P 〉 0.05 ）. Ubiquitinated proteins of 50 000- 250 000 increased after the oxidative stress, but if RPE cells were preconditioned by MG-132, ubiquitinated proteins increased mainly over 75 000. Ubiquitinated protein expressions in cells increased after treating mainly in the nucleus. Conclusion Ubiquitination is induced in ARPE-19 cells by oxidative stress in vitro.