贝伐单抗对视网膜色素上皮细胞抗氧化功能的影响

发布时间：2018-02-28 20:40

本文关键词： 年龄相关性黄斑变性贝伐单抗活性氧人视网膜色素上皮细胞　出处：《眼科新进展》2017年04期 　论文类型：期刊论文

【摘要】：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0 h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,0 h组、12 h组、24 h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(P0.05);与对照组相比,12 h、24 h、48 h、72 h组细胞内ROS水平上升,差异有统计学意义(P0.05);线粒体膜电位在12 h、24 h、48 h、72 h组均较对照组降低,差异有统计学意义(P0.01),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、24 h、48 h和72 h组均上升,而且在24 h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(P0.01)。与对照组比较,HO-1 mRNA的表达在24 h、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(P0.05)。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。
[Abstract]:Objective to observe the effect of bevacizumab on the antioxidant function of human retinal pigment epithelium (RPE) cells. To explore the possible mechanism of anti vascular endothelial growth factor (VEGF) vascular endothelial growth factor (VEGF) preparation in the treatment of macular atrophy after age-related macular degeneration. Methods Human RPE cell line ARPE-19 cells were cultured with DMED/F12 medium containing 0.25g 路L ~ (-1) final concentration bevacizumab. According to the time of treatment, 5 groups were divided into 0 h group (control group, 12 h group, 24 h group, 48 h group, 72 h group). Oxidative stress was induced by H _ 2O _ 2.MitoSox Red fluorescence staining was used to detect the mitochondrial reactive oxygen species (Ros) production level and JC-1 fluorescence staining to detect the change of mitochondrial membrane potential. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of NADPH oxidase 4 (NADPH oxidase 4NOX4) and heme oxygenase-1 (HO-1HO-1) in each group. Results the results of CCK-8 were significant. The results showed that there was no significant difference in cell activity between 0 h group and 12 h group and 24 h group and 72 h group were 100.2 卤3.3 h and 99.2 卤2.7%, respectively. There was no significant difference between the two groups (103.9 卤3.7% and 103.6 卤3.3%), and the level of ROS was increased compared with the control group. The mitochondrial membrane potential decreased at 12 h, 24 h, 48 h and 72 h compared with the control group, and the difference was statistically significant at 48 h and the lowest at 72 h, and the mitochondrial membrane potential was significantly higher than that in the control group at 48 h. However, the results of RT-PCR and Western blot showed that the expression of nox4 mRNA and protein increased at 12 h, 24 h and 72 h, and reached the highest level at 24 h, then decreased significantly, but still higher than that in control group. Compared with the control group, the expression of HO-1 mRNA decreased at 24 h and 72 h, while the expression of HO-1 protein decreased at 48 h and 72 h. Conclusion the clinical concentration of bevacizumab can reduce the antioxidant function of RPE, which may be one of the causes of macular progressive atrophy after long-term anti VEGF therapy.
【作者单位】：青岛大学医学院、山东省眼科研究所青岛眼科医院;山东省眼科研究所山东省眼科学重点实验室;山东省眼科研究所青岛眼科医院;
【基金】：国家自然科学基金面上项目(编号:81670828) 山东省自然科学基金资助(编号:ZR2014HM029)~~
【分类号】：R774.5

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