贝伐单抗对人视网膜色素上皮细胞抗氧化功能的影响

发布时间：2019-01-09 19:15

【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:采用人视网膜色素上皮细胞系ARPE-19细胞,培养液中加入终浓度为0.25 g·L-1贝伐单抗,根据不同的处理时间分为0h组(对照组)、12h组、24h组、48h组、72h组共5组,加入H2O2诱导氧化应激反应。采用CCK-8法检测细胞活性,用MitoSox Red荧光染色检测细胞内线粒体活性氧(mitochondrialreactive oxygen species,mtROS)产生水平,通过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)检测促氧化因子NADPH氧化酶4(NADPH oxidase 4,NOX4),抗氧化因子血红素氧合酶-1(Heme oxygenase-1,HO-1)、NAD(P)H醌氧化还原酶-1(NAD(P)H quinone oxidoreductase,NQO-1)和超氧化物歧化酶2(superoxide dismutase 2,SOD2)基因和蛋白的表达水平。结果:CCK-8检测结果显示:上述处理对细胞活性无显著影响,0h组、12h组、24h组、48h组和72h组细胞活性分别为(100.2±3.3)%、(99.2±2.7)%,(102.5±6.4)%、(103.9±3.7)%、(103.6±3.3)%,差异无统计学意义(P0.05);细胞内线粒体ROS的检测结果显示:与对照组相比,12h、24h、48h、72h组细胞内线粒体ROS水平均上升,差异有统计学意义(P0.05);线粒体膜电位检测结果显示:线粒体膜电位在12h、24h、48h、72h组均较对照组降低,差异有显著性,48h达最低,72h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基因和蛋白的相对表达量在12h、24h、48h和72h组均明显上升,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(P0.01)。与对照组比较,HO-1基因的相对表达量在24h、48h和72h组均明显下降,而HO-1蛋白的表达在48h和72h组均下降,差异有统计学意义(P0.05)。与对照组比较,NQO-1基因和蛋白的相对表达量在24h、48h和72h组均明显下降,差异有统计学意义(P0.05)。与对照组比较,SOD2基因和蛋白的表达在12h、24h、48h和72h组均未见明显变化,差异无统计学意义(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 (retinal pigment epithelium,RPE) cells and to explore the anti-neovascularization endothelial growth factor (vascular endothelial growth factor,). The possible mechanism of VEGF in the treatment of macular atrophy after age-related macular degeneration. Methods: human retinal pigment epithelial cell line ARPE-19 cells were used. The final concentration of bevacizumab was 0.25 g / L in the culture medium. According to different treatment time, the cells were divided into 0 h group (control group), 12 h group, 24 h group and 48 h group. In 72 h group, 5 groups were added H2O2 to induce oxidative stress reaction. CCK-8 assay was used to detect cell activity, MitoSox Red fluorescence staining was used to detect the level of reactive oxygen species (mitochondrialreactive oxygen species,mtROS) production in mitochondria, and JC-1 fluorescence staining was used to detect the change of mitochondrial membrane potential. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot (Western blot) were used to detect the oxidant factor NADPH oxidase 4 (NOX4) and the antioxidant factor heme oxygenase-1 (Heme oxygenase-1,HO-1), respectively. The expression levels of NAD (P) H quinone redox enzyme 1 (NAD (P) H quinone oxidoreductase,NQO-1 and superoxide dismutase 2 (superoxide dismutase 2 so 2) gene and protein. Results: the results of CCK-8 showed that the cell activity was (100.2 卤3.3)%, (99.2 卤2.7)%, (102.5 卤6.4)% in 0 h group, 12 h group, 24 h group, 48 h group and 72 h group, respectively. (103.9 卤3.7)%, (103.6 卤3.3)%, the difference was not statistically significant (P0.05); The results of intracellular mitochondrial ROS detection showed that: compared with the control group, the level of mitochondrial ROS increased significantly in 12h / 24h / 48h / 72h group (P0.05). The results of mitochondrial membrane potential detection showed that the mitochondrial membrane potential was significantly lower than that of the control group at 12 h, 24 h, 48 h and 72 h, which was the lowest at 48 h and increased significantly at 72 h, but still lower than that in the control group. The results of RT-PCR and Western blot analysis showed that the relative expression of NOX4 gene and protein increased significantly at 12h, 24h, 48h and 72h, and reached the highest level at 24h, then decreased significantly, but still higher than that of control group. The difference was statistically significant (P0.01). Compared with the control group, the relative expression of HO-1 gene decreased significantly at 24 h, 48 h and 72 h, while the expression of HO-1 protein decreased at 48 h and 72 h, the difference was statistically significant (P0.05). Compared with the control group, the relative expression of NQO-1 gene and protein decreased significantly at 24 h, 48 h and 72 h, the difference was statistically significant (P0.05). Compared with the control group, the expression of SOD2 gene and protein did not change significantly at 12h, 24h, 48h and 72h, the difference was not statistically significant (P0.05). 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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774

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