摘要目的:观察聚嘧啶束结合蛋白相关剪接因子（PSF）高表达对高浓度4-羟基壬烯醛（4-HNE）诱导下人视网膜微血管内皮细胞（hRMEC）内质网（ER）氧化应激损伤的保护作用。方法:体外培养的对数生长期hRMEC分为正常组、单纯4-HNE处理组（单纯4-HNE组），空载质粒联合4-HNE处理组（Vec+4-HNE组）、PSF高表达联合4-HNE处理组（PSF+ 4-HNE组）。单纯4-HNE组、Vec+4-HNE组、PSF+4-HNE组细胞培养基加入10 μmol/L 4-HNE，刺激12 h。其后，Vec+4-HNE组、PSF+4-HNE组应用转染试剂脂质体2000分别导入pcDNA空载体、pcDNA-PSF真核表达质粒，转染24 h。正常组细胞常规培养。流式细胞仪检测各组细胞凋亡率；2'，7'-二氯二氢荧光素双乙酸盐探针检测各组细胞内活性氧（ROS）表达水平。蛋白质免疫印迹法检测各组细胞内ER氧化物蛋白1（Ero-1）、蛋白质二硫键异构酶（PDI）、C/EBP同源转录因子（CHOP）、葡萄糖调节蛋白（GRP）78、蛋白激酶R样ER激酶（pERK）/磷酸化pERK（p-pERK）、真核起始因子（eIF）2α/磷酸化eIF（peIF）、活化转录因子4（ATF4）蛋白相对表达量。组间比较行单因素方差分析。结果:单纯4-HNE组、Vec+4-HNE组、PSF+4-HNE组细胞凋亡率分别为（22.50±0.58）%、（26.93±0.55）%、（11.70±0.17）%；细胞内ROS表达量分别为0.23±0.03、1.60±0.06、0.50±0.06。三组间细胞凋亡率比较，差异有统计学意义（ F=24.531， P<0.05）；Vec+4-HNE组ROS表达水平较单纯4-HNE组、PSF+4-HNE组显著升高，差异有统计学意义（ F=37.274， P<0.05）。正常组、单纯4-HNE组、Vec+4-HNE组、PSF+4-HNE组细胞ER中Ero-1、PDI蛋白相对表达量分别为1.25±0.03、0.45±0.03、0.63±0.03、1.13±0.09和1.00±0.10、0.27±0.10、0.31±0.05、0.80±0.06；CHOP、GRP78蛋白相对表达量分别为0.55±0.06、1.13±0.09、0.90±0.06、0.48±0.04和0.48±0.04、1.25±0.03、1.03±0.09、0.50±0.06。4组Ero-1（ F=43.164）、PDI（ F=36.643）、CHOP（ F=42.855）、GRP78（ F=45.275）蛋白相对表达量比较，差异均有有统计学意义（ P<0.05）。4组细胞ER p-pERK/pERK（ F=35.755）、peIF2α/eIF（ F=38.643）、ATF4（ F=31.275）蛋白相对表达量比较，差异均有统计学意义（ P<0.05）。 结论:PSF可抑制高浓度4-HNE诱导的细胞凋亡及ROS产生；其作用机制与恢复ER稳态平衡及下调pERK/eIF2α/ATF4通路的活化水平密切相关。

abstractsObjective:To observe the effects of overexpression of polypyrimidine tract binding protein-associated splicing factor (PSF) on the endoplasmic reticulum (ER) oxidative stress damage of human retinal microvascular endothelial cells (hRMEC) under high concentration of 4-hydroxynonenal (4-HNE).Methods:The logarithmic growth phase hRMEC cultured in vitro was divided into normal group, simple 4-HNE treatment group (simple 4-HNE group), empty plasmid combined with 4-HNE treatment group (Vec+4-HNE group), and PSF high expression combined with 4-HNE treatment group (PSF+4-HNE group). In 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group cell culture medium, 10 μmol/L 4-HNE was added and stimulated for 12 hours. Subsequently, the Vec+4-HNE group and PSF+4-HNE group were transfected with transfection reagent liposome 2000 into pcDNA empty bodies and pcDNA-PSF eukaryotic expression plasmids, respectively, for 24 hours. Flow cytometry was used to detect the effects of 4-HNE and PSF on cell apoptosis. The effect of PSF overexpression on the expression of reactive oxygen species (ROS) in hRMEC was detected by 2', 7'-dichlorodihydrofluorescein double Acetate probe. Western blot was used to detect ER oxide protein 1 (Ero-1), protein disulfide isomerase (PDI), C/EBP homologous transcription factor (CHOP), glucose regulatory protein (GRP) 78, protein kinase R-like ER kinase (PERK)/phosphorylated PERK (p-PERK), and Eukaryotic initiation factor (eIF) 2α/the relative expression levels of phosphorylated eIF (peIF) and activated transcription factor 4 (ATF4) proteins in hRMEC of normal group, 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group. Single factor analysis of variance was performed for inter group comparison.Results:The apoptosis rates of the simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were (22.50±0.58)%, (26.93±0.55)%, and (11.70±0.17)%, respectively. The intracellular ROS expression levels were 0.23±0.03, 1.60±0.06, and 0.50±0.06, respectively. The difference in cell apoptosis rate among the three groups was statistically significant ( F=24.531, P<0.05). The expression level of ROS in the Vec+4-HNE group was significantly higher than that in the simple 4-HNE group and the PSF+4-HNE group, with a statistically significant difference ( F=37.274, P<0.05). The relative expression levels of ER Ero-1 and PDI proteins in the normal group, simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were 1.25±0.03, 0.45±0.03, 0.63±0.03, 1.13±0.09, and 1.00±0.10, 0.27±0.10, 0.31±0.05, and 0.80±0.06, respectively. The relative expression levels of CHOP and GRP78 proteins were 0.55±0.06, 1.13±0.09, 0.90±0.06, 0.48±0.04 and 0.48±0.04, 1.25±0.03, 1.03±0.09, 0.50±0.06, respectively. The relative expression levels of Ero-1 ( F=43.164), PDI ( F=36.643), CHOP ( F=42.855), and GRP78 ( F=45.275) proteins in four groups were compared, and the differences were statistically significant ( P<0.05). Four groups of cells ER p-pERK/pERK ( F=35.755), peIF2 α/ The relative expression levels of eIF ( F=38.643) and ATF4 ( F=31.275) proteins were compared, and the differences were statistically significant ( P<0.05). Conclusion:PSF can inhibit cell apoptosis and ROS production induced by high concentration of 4-HNE, and its mechanism is closely related to restoring the homeostasis of ER and down-regulating the activation level of PERK/eIF2α/ATF4 pathway.