聚嘧啶束结合蛋白相关剪接子高表达对人视网膜微血管内皮细胞凋亡和内质网氧化应激损伤的保护作用  

Protective effect of polypyrimidine tract-binding protein-associated splicing factor on endoplasmic reticulum oxidative stress injury of human retinal microvascular endothelial cells

作　　者：李文博 曹靖靖 寇振宇 韩菲菲 刘爱华 东莉洁 Li Wenbo;Cao Jingjing;Kou Zhenyu;Han Feifei;Liu Aihua;Dong Lijie(Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin Branch of National Clinical Research Center for Ocular Disease,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院、眼视光学院、眼科研究所、国家眼耳鼻喉疾病临床医学研究中心、天津市分中心、天津市视网膜功能与疾病重点实验室,天津300384

出　　处：《中华眼底病杂志》2023年第8期681-686,共6页Chinese Journal of Ocular Fundus Diseases

基　　金：天津市高等教育委员会科技发展基金项目(2022ZD057)。

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-羟基王烯醛(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组细胞培养基加人10μmol/L4-HNE,刺激12h。其后,Vec+4-HNE组、PSF+4-HNE组应用转染试剂脂质体2000分别导人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1.000.10、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ERp-pERK/pEObjective 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,10μmol/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(elF)2a/the relative expression levels of phosphorylated eIF(pelF)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 R0S 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 l

关 键 词：聚嘧啶束结合蛋白相关剪接子 高浓度4-羟基壬烯醛 内质网 人视网膜血管内皮细胞 细胞实验 

分 类 号：R774.1[医药卫生—眼科]