摘要
目的观察还原型烟酰胺腺嘌呤二核苷酸氧化酶4(NOX4)抑制剂对缺氧诱导的人视网膜色素上皮(RPE)细胞中血管内皮生长因子(VEGF)表达的影响。方法将APRE-19细胞分为Avastin干预组和VAS2870干预组,并按照药物剂量不同将Avastin干预组亚分为常氧对照组、缺氧对照组及0.25 mg/ml、0.50 mg/ml和0.75 mg/ml Avastin干预组,将VAS2870干预组亚分为1μmol/ml、3μmol/ml和5μmol/ml VAS2870干预组,缺氧对照组采用终浓度为300μmol/L CoCl2处理ARPE-19细胞以建立细胞化学缺氧模型。采用细胞免疫荧光染色技术对不同干预组细胞中NOX4和VEGF表达进行测定和定位,采用Western blot法对不同干预组细胞中NOX4和VEGF蛋白相对表达量进行测定和比较。结果Western blot法检测显示,常氧对照组、缺氧对照组及0.25 mg/ml、0.50 mg/ml和0.75 mg/ml Avastin干预组NOX4蛋白相对表达量分别为0.657±0.153、1.000±0.200、1.206±0.300、1.260±0.200和1.413±0.273,VEGF-A蛋白相对表达量分别为0.821±0.110、1.210±0.100、0.672±0.100、0.340±0.120和0.300±0.130,组间总体比较差异均有统计学意义(F=17.631,P<0.001;F=4.777,P=0.020),其中0.75 mg/ml Avastin干预组细胞中NOX4蛋白表达量明显高于常氧对照组,差异有统计学意义(P<0.001),0.25、0.50和0.75 mg/ml Avastin干预组VEGF-A表达量均明显低于缺氧对照组,差异均有统计学意义(均P<0.05)。常氧对照组、缺氧对照组及1μmol/ml、3μmol/ml和5μmol/ml VAS2870干预组细胞中NOX4的蛋白相对表达量分别为0.970+0.120、1.060+0.130、0.880+0.130、0.567+0.135和0.450+0.120,VEGF-A蛋白相对表达量分别为0.387+0.135、0.627+0.125、0.370+0.140、0.363+0.140和0.160+0.100,组间总体比较差异均有统计学意义(F=12.933,P<0.001;F=4.948,P<0.05),其中3μmol/ml和5μmol/ml VAS2870干预组细胞中NOX4蛋白相对表达量明显低于缺氧对照组,差异均有统计学意义(均P<0.001);1μmol/ml、3μmol/ml和5μmol/ml VAS2870干预组细胞中VEGF-A蛋白
Objective To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)on vascular endothelial growth factor(VEGF)expression in hypoxia-induced human retinal pigment epithelial cells.Methods The cultured APRE-19 cells were divided into Avastin group and VAS2870 group,and then the Avastin group was subdivided into the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,and the VAS2870 group was subdivided into 1μmol/ml VAS2870 intervention group,3μmol/ml VAS2870 intervention group and 5μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining,and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.Results The relative expression of NOX4 was 0.657±0.153,1.000±0.200,1.206±0.300,1.260±0.200 and 1.413±0.273,and the relative expression of VEGF-A was 0.821±0.110,1.210±0.100,0.672±0.100,0.340±0.120 and 0.300±0.130 in the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,respectively,with statistically significant differences among the groups(F=17.631,P<0.001;F=4.777,P<0.05).The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group(P<0.001).The relative expression of VEGF-A protein in the cells of the 0.25,0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group(P<0.05).The expression of NOX4 protein in the cells was 0.970±0.120,1.060±0.130,0.880±0.130,0.567±0.135 and 0.450±0.120,and the relative expression of VEGF-A protein was 0.387±0.135,0.627±0.125,0.370±0.140,0
作者
李静
杨静
康前雁
Li Jing;Yang Jing;Kang Qianyan(Department of Ophthalmology,Shanxi Provincial People's Hospital,Xi'an 710068,China;Department of Ophthalmology,The First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2020年第1期4-9,共6页
Chinese Journal Of Experimental Ophthalmology
基金
陕西省重点研发计划项目(2018SF-155)
西安市科技计划项目(2016051SF/YX07)。
