贝伐单抗对视网膜色素上皮细胞抗氧化功能的影响

目的 观察贝伐单抗对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞抗氧化功能的影响,以探讨抗新生血管内皮生长因子(vascular endothelial growth factor,VEGF)制剂治疗年龄相关性黄斑变性后黄斑部萎缩的可能机制.方法 用含终浓度0.25g·L-1贝伐单抗的DMED/F12培养液培养人RPE细胞系ARPE-19细胞,根据处理时间的不同分为Oh组(对照组)、12 h组、24 h组、48 h组、72 h组共5组,加入H2O2诱导氧化应激反应.用CCK-8法检测细胞活性,MitoSox Red荧光染色检测细胞内线粒体活性氧(reactive oxygen species,ROS)产生水平,JC-1荧光染色检测细胞线粒体膜电位的变化;分别用逆转录聚合酶链反应(RT-PCR)及免疫蛋白印迹法(Western blot)检测各组促氧化因子NADPH氧化酶4(NADPH oxidase 4,NOX4)和抗氧化因子血红素氧合酶-1(heme oxygenase 1,HO-1) mRNA和蛋白的表达水平.结果 CCK-8检测结果显示:上述处理对细胞活性无显著影响,Oh组、12 h组、24h组、48h组和72 h组细胞活性分别为(100.2±3.3)％、(99.2±2.7)％、(102.5±6.4)％、(103.9±3.7)％和(103.6±3.3)％,差异无统计学意义(P＞0.05);与对照组相比,12 h、24h、48 h、72 h组细胞内ROS水平上升,差异有统计学意义(P＜0.05);线粒体膜电位在12 h、24h、48 h、72 h组均较对照组降低,差异有统计学意义(P＜0.01),48 h达最低,72 h时显著提高,但仍低于对照组.RT-PCR和Western blot检测结果显示:与对照组比较,NOX4 mRNA和蛋白的表达在12 h、24h、48 h和72 h组均上升,而且在24h表达最高,之后明显下降,但仍高于对照组,差异有统计学意义(P＜0.01).与对照组比较,HO-1 mRNA的表达在24h、48 h和72 h组均下降,而HO-1蛋白的表达在48 h和72 h组下降,差异有统计学意义(P＜0.05).结论 临床浓度的贝伐单抗可以降低RPE的抗氧化功能,可能是长期抗VEGF治疗后黄斑部进行性萎缩的原因之一.

Effects of bevacizumab on antioxidative function in human retinal pigment epithelial cells

Objective To investigate the effects of bevacizunab on the antioxidative function of human retinal pigment epithelium (RPE),in order to explore the possible mechanism of macular atrophy induced by the application of anti-vascular epithelial growth factor (VEGF) agents in age-related macular degeneration.Methods Human RPE cells were incubated in DMEM/F12 medium containing 0.25 g · L-1 bevacizumab and divided into 5 groups according to incubation period:0 hour(control),12 hours,24 hours,48 hours and 72 hours,and then the oxidative stress was induced by adding H2O2.Cell viability was measured by the CCK8 assay.MitoSox Red was used to determine mitochondrial reactive oxygen species (mtROS) production.Mitochondrial membrane potential was measured using the JC-1 assay.The expression levels of NOX4 and HO-1 were detected by RT-PCR and Western blot,respectively.Results CCK8 assay determination showed that the above treatment had no significant effect on cell viability,the cell viability of 0 hours,12 hours,24 hours,48 hours and 72 hours were (100.2 ±3.3)％,(99.2 ±2.7)％,(102.5 ±6.4)％,(103.9 ±3.7)％,(103.6 ±3.3)％,the difference was not statistically significant (P ＞ 0.05).Compared with the control group,the levels of mtROS increased at 12 hours,24 hours,48 hours and 72 hours,the difference was statistically significant (P ＜ 0.05).Mitochondrial membrane potential at 12 hours,24 hours,48 hours,72 hours were lower than the control group,the difference was significant,48 hours reached the lowest,72 hours significantly increased,but still lower than the control group.RT-PCR and western blot results demonstrated that the expression of NOX4 mRNA and protein increased at 12 hours,24 hours,48 hours and 72 hours,and reached the highest at 24 hours,then decreased significantly,but still higher than the control group,the difference was statistically significant (P ＜0.01).Compared with the control group,the expression of HO-1 mRNA decreased at 24 hours,48 hours and 72 hours,while the expression of HO-1 protein decreased at 48 hours and 72 hours,the difference was statistically significant (P ＜ 0.05).Conclusion The clinical concentration of bevacizumab can reduce the anti-oxidative function of RPE cells,which may be one of the causes of progressive macular atrophy after long-term anti-VEGF therapy.