p75NTR受体过表达对人RPE细胞氧化应激损伤的促进作用

Promoting effect of p75NTR receptor overexpression on oxidative stress injury in human retinal pigment epithelium cells

背景脉络膜新生血管（CNV）是多种眼底疾病致盲的病理基础，视网膜色素上皮（RPE）细胞氧化应激损伤在CNV生成中具有重要作用。RPE氧化应激损伤能激活肿瘤坏死因子超家族的p75NTR受体，引起血管内皮细胞增生，但其调控机制尚未明确。 目的探讨RPE细胞中p75NTR受体在CNV形成过程中的作用，并对其作用机制进行分析。 方法体外培养人RPE细胞（ARPE19细胞系），用p75NTR受体过表达质粒转染RPE细胞，分别采用逆转录PCR（RT-PCR）和Western blot法进行转染细胞中p75NTR受体基因和蛋白水平检测，以未转染p75NTR受体过表达质粒的RPE细胞作为对照组。采用BrdU法检测各组细胞的增生活性；采用Annexin V-FITC/PI双染法及流式细胞仪检测各组细胞的凋亡率；用H2DCFDA荧光法和流式细胞计数检测细胞内活性氧簇（ROS）阳性百分数；采用激光扫描共焦显微镜观察各组细胞线粒体标志物的表达（膜电位）及细胞色素C的表达；采用Western blot法检测和比较各组细胞中裂解caspase-3、Fas及血管内皮生长因子165（VEGF165）蛋白的相对表达水平。 结果p75NTR受体质粒转染组细胞中p75NTR受体的mRNA及蛋白表达量分别是对照组的（6.11±0.77）倍和（7.42±0.48）倍，差异均有统计学意义（t＝11.49、23.17，均P〈0.01）。BrdU法检测结果显示，p75NTR受体质粒转染12、24、36和48 h组细胞的增生活性（A值）率分别为（93.12±0.56）%、（86.30±0.66）%、（72.53±0.86）%和（60.77±2.81）%，对照组为100%，随着p75NTR受体质粒转染时间的延长，RPE细胞活性值逐渐减低，细胞凋亡百分数明显增加，与对照组比较差异均有统计学意义（均P〈0.05）。p75NTR受体质粒转染组转染后48 h，RPE细胞内ROS相对荧光强度为对照组的2.4倍，差异有统计学意义（t＝16.45，P〈0.01）；对照组RPE细胞中线粒体标志物阳性细胞比例为100%，而p75NTR受体质粒转染组细胞中线粒体标志物阳性细胞比例为（37.30±2.06）%，2个组比较差异有统计学意义（t＝57.71，P〈0.01）。p75NTR受体质粒转染组RPE细胞中细胞色素C的荧光强度明显高于对照组，与对照组比较，p75NTR受体质粒转染组RPE细胞中裂解caspase-3、Fas及VEGF165蛋白表达水平明显升高，差异均有统计学意义（均P〈0.01）。 结论p75NTR受体过表达引起RPE细胞线粒体损伤及细胞凋亡，同时刺激RPE细胞分泌血管生成因子，促进CNV的形成。p75NTR受体可能是调控RPE损伤的另一个重要通路。

BackgroundChoroidal neovascularization （CNV） is the primary pathogenic cause of many fundus diseases.Oxidative stress injury of retinal pigment epithelial （RPE） cells plays important role in angiogenesis of choroid new blood vessels.Oxidative stress injury can active p75NTR receptor, a member of tumor necrosis factors family, resulting in the proliferation of vascular endothelial cells.However, the mechanisms of vascular endothelial cell proliferation remain unclear. ObjectiveThis study was conducted to investigate the effect of p75NTR overexpression on CNV and the relative mechanism. MethodsThe ARPE-19 cell line was used in this study.RPE cells were transfected with p75 NTR receptor overexpressed plasmid, and untransfected cells served as the control group.The transfected results were verified by reverse transcription-PCR and Western blot assay.Viability of the cells over time was determined in the p75 NTR receptor plasmid transfected group by using BrdU assay.The percentage of apoptotic cells was detected by flow cytometry using Annexin V-FITC/PI fluorescence staining.The percentage of reactive oxygen species （ROS） expression in the cells was detected by using H2DCFDA fluorescence and flow cytometry.Mitochondrial membrane potential and cytochrome C expression were examined under the confocal microscope.The protein expressions of cleaved caspase-3, Fas and VEGF were determined by Western blot assay. ResultsThe relative expression level of p75NTR receptor mRNA was （6.11±0.77） times higher than that of the control group, and relative expression level of p75NTR receptor protein in the cells in the p75 NTR receptor plasmid transfected group was （7.42±0.48） times higher than that in the control group （t=11.49 and 23.17, both at P〈0.01）. The absorbency values of the p75NTR receptor plasmid transfected group were （93.12±0.56）%, （86.30±0.66）%, （72.53±0.86）% and （60.77±2.81）% in 12, 24, 36 and 48 hours after plasmid transfection, which were significantly lower than 100% in the control group, and the apoptotic percentages were evidently higher than that in the control group （all at P〈0.05）. The relative fluorescence intensity of ROS fluorescence in the p75NTR receptor plasmid transfected group was 2.4 times higher than that in the control group, showing significant difference （t=16.45, P〈0.01）. The positive expressing rate of mitomarker （mitochondrial membrane potentials） was 100% in the control group and （37.30±2.06）% in the p75NTR receptor plasmid transfected group, with significant difference between them （t=57.71, P〈0.01）. The fluorescence intensity of cytochrome C expression was elevated in the p75NTR receptor plasmid transfected group compared with the control group.Compared with the control group, the expressing levels of cleaved caspase-3, Fas and VEGF165 proteins in the cells were significantly raised in the p75NTR receptor plasmid transfected group （all at P〈0.01）. ConclusionsOverexpression of p75 NTR receptors in RPE cells leads to mitochondrial damage and cellular apoptosis and the secretion of VEGF protein, which sequentially promote CNV.P75 NTR receptor may be another important regulation pathway in RPE oxygen damage.