蛋白激酶C活化对兔视网膜色素上皮细胞抗氧化能力的影响及机制研究  

Influence of Protein kinase C activation on antioxidant capacity of rabbit retinal pigment epithelium cells and its mechanisms

作　　者：卫冬 陈丽娟 教莹莹 李红玉 孙兆义 WEI Dong;CHEN Li-juan;JIAO Ying-ying;LI Hong-yu;SUN Zhao-yi(The Third Department of Ophthalmology, Hongqi Hospital Affiliated to Mudanjiang Medical University, Heilongjiang Province, Mudanjiang 157000, China;The Second Department of Ophthalmology, Hongqi Hospital Affiliated to Mudanjiang Medical University, Heilongjiang Province, Mudanjiang 157000, China;Department of Ophthalmology,Mudanjiang Medical University, Heilongjiang Province, Mudanjiang 157000, China)

机构地区：[1]牡丹江医学院附属红旗医院眼三科,黑龙江牡丹江157000 [2]牡丹江医学院附属红旗医院眼二科,黑龙江牡丹江157000 [3]牡丹江医学院眼科,黑龙江牡丹江157000

出　　处：《中国当代医药》2018年第33期16-20,共5页China Modern Medicine

基　　金：黑龙江省卫生计生委科研课题(2016-379)

摘　　要：目的通过激活兔视网膜色素上皮细胞(RPE)内蛋白激酶C(PKC),观察RPE细胞核内转录因子E2相关因子2(Nrf2)的表达情况以及细胞内谷胱甘肽(GSH)的表达水平。方法应用免疫荧光染色鉴定原代培养兔视网膜色素上皮细胞(RPE),将第3代RPE细胞分为空白对照组(15%胎牛血清的DMEM培养液,n=8)、PMA组(15%胎牛血清的DMEM培养液+4μl 100 nmol/L PKC特异激活剂佛波酯溶液,n=8)和PMA+BST组(用4μl 10μmol/L Nrf2抑制剂鸦胆苦醇溶液预处理RPE细胞1 h后,吸出并加入含PMA 4μl 100 nmol/L的15%胎牛血清的DMEM培养液,n=8),培养24 h后,采用Western-blot方法检测Nrf2蛋白在胞质和胞核内的表达,采用GSH试剂盒检测各组RPE细胞内GSH的表达,对细胞核内Nrf2及细胞内GSH表达进行相关性分析,采用免疫荧光染色检测Nrf2蛋白在细胞内转位情况。结果 RPE细胞胞质内可见黑色素颗粒,呈长梭形,鼠抗人角蛋白(AE1/AE3)免疫荧光染色鉴定呈强阳性;Western blot检测结果显示,与对照组比较,PMA组和PMA+BST组胞质内Nrf2蛋白表达显著减少,胞核内Nrf2蛋白表达显著增加,其中PMA组胞质内Nrf2蛋白表达较PMA+BST组显著降低,而细胞核内Nrf2蛋白表达则显著增多(胞质:F=26.62,P<0.05;胞核:F=29.88,P<0.05)。GSH检测试剂盒检测显示,与对照组比较,PMA组和PMA+BST组细胞内GSH表达显著增高,其中PMA组细胞内GSH表达显著高于PMA+BST组(F=37.64,P<0.05)。细胞核内Nrf2与细胞内GSH表达成正相关(R2=0.908,P<0.05)。免疫荧光染色结果显示,与对照组相比,PMA组和PMA+BST组细胞核内Nrf2蛋白表达增加,发生了核转位,其中PMA组较PMA+BST组核转位更为显著。结论 PKC活化能够促进RPE细胞抗氧化能力,其作用机制与PKC/Nrf2信号通路调节有关。Objective To observe the expression status of nuclear factor erythroid2-related factor2(Nrf2)and glu-tathione(GSH)in retinal pigment epithelium(RPE)cells following the activation of protein kinase C(PKC)in rabbits.Methods Immunofluorescence staining was used to identify the original generation cultured RPE cells in rabbits.The third generation of RPE cells was divided into the control group(DMEM culture medium contains15%fetal bovine serum,n=8),the PMA group(DMEM culture medium contains15%fetal bovine serum+4μl100nmol/L PKC specific activator phorbol ester solution,n=8)and the PMA+BST group(pretreatment of RPE cells with4μl of10μmol/L Nrf2inhibitor brusatol solution[BST]for an hour,aspirate and add DMEM culture medium containing PMA4μl of100nmol/L and15%fetal bovine serum,n=8),the incubation time of each group was24hours,Western-blot was used to detect the expression of Nrf2protein in cytoplasm and nucleus.Then the GSH kit was used to detect the ex-pression of GSH in RPE cells.And the correlation be-tween the expression of Nrf2in the nucleus and intra-cellular GSH was analyzed.Immunofluorescence staining was used to detect the translocation of Nrf2protein in the cells.Results RPE cells showed melanin granules in the cytoplasm,long spindle-shape,and anti-human keratin(AE1/AE3)immunofluorescence staining showed strong positive.Western blot showed that the expression of Nrf2protein in cell cytoplasm of the PMA group and the PMA+BST group were significantly down regulated compared with the control group,while the Nrf2protein expression in cell nucleus was markedly up-regulated(all P<0.05).Moreover,the differ-ence in the expression of Nrf2in cytoplasm and nucleus between the three groups was statistically significant(cyto-plasm:F=26.62,P<0.05;nucleus:F=29.88,P<0.05).GSH kit showed that,when compared with the control group,the intracellular expression levels of GSH in the PMA group and the PMA+BST group were significantly increased(all P<0.05),and the GSH expression level of the PMA group was significantly higher t

关 键 词：视网膜色素上皮细胞 蛋白激酶C 核因子E2相关因子2 谷胱甘肽 

分 类 号：R332[医药卫生—人体生理学]