摘要
目的:观察内质网应激(ERS)在氧化低密度脂蛋白(OxLDL)诱导的人视网膜色素上皮(RPE)细胞凋亡中的作用。方法:人RPE细胞系ARPE19采用低糖DMEM培养基和10%胎牛血清进行常规培养。实验分为3组:对照组(常规培养的ARPE19)、OxLDL组(加入5、10、25、50、100μg/mL OxLDL)和LDL组(加入5、10、25、50、100μg/mL LDL)培养24h。分别采用细胞计数试剂盒(CCK8)检测各组细胞活性,流式细胞仪检测凋亡比例,蛋白质印迹(Western blotting)检测ERS相关蛋白及凋亡相关酶的表达。激光共聚焦显微镜观察人RPE细胞吞噬红色荧光探针Dil标记的氧化低密度脂蛋白(Dil-OxLDL)情况。结果:CCK8结果显示:对照组细胞存活率为(100±5.637)%,加入5、10、25、50、100μg/mL OxLDL后细胞活力分别为(105.298±9.395)%、(97.106±5.417)%、(77.015±4.055)%、(67.613±3.853)%和(43.872±9.532)%(P<0.05);加入5、10、25、50、100μg/mL LDL后细胞活力分别为(97.55±6.217)%、(99.640±3.586)%、(90.495±2.786)%、(83.552±9.171)%和(90.910±1.429)%(P>0.05)。流式细胞仪结果显示浓度为25μg/mL的OxLDL会明显诱导细胞凋亡,对照组、OxLDL组(25μg/mL)和LDL(25μg/mL)组凋亡率分别是(5.271±0.519)%、(41.23±1.686)%和(13.07±2.579)%(P<0.01);Western blotting结果显示OxLDL(25μg/mL)组的ERS相关蛋白和凋亡相关酶的表达量明显高于对照组与LDL组(Caspase-12:F=50.53,P<0.05;GRP78:F=55.60,P<0.05;CHOP:F=38.22,P<0.05;XBP-1:F=53.94,P<0.05;ATF6:F=20.01,P<0.05),而LDL组(25μg/mL)和对照组之间无差异(P>0.05)。结论:ERS参与了OxLDL诱导的人RPE细胞凋亡,调控ERS可能抑制人RPE细胞凋亡,从而治疗RPE细胞凋亡相关疾病。
AIM:To investigate the role of endoplasmic reticulum stress(ERS)in oxidized low-density lipoprotein(OxLDL)induced retinal pigment epithelium(RPE)cells apoptosis.METHODS:The human RPE cell line ARPE19 was cultured in low glucose DMEM medium with 10%fetal bovine serum.Experiments were divided into three groups:control group(normal cultured ARPE19),OxLDL group(with 5,10,25,50,100μg/mL of OxLDL),and LDL group(with 5,10,25,50,100μg/mL of OxLDL)and cultured 24h.The CCK8 kit(Cell Counting Kit-8)was used to detect cell activity,the flow cytometry was used to detect the percentage of apoptosis and the Western blotting was used to detect the expression of ERS-related proteins and apoptosis-related enzymes.The confocal microscope was used to observe the phagocytosis of Dil-labeled OxLDL(Dil-OxLDL)in RPE cells.RESULTS:The results of CCK8 showed that when compared with control group,with cell viability of(100±5.637)%,different concentrations(5,10,25,50,100μg/mL)of OxLDL could change cell viability significantly(F=41.20,P<0.05),and cell viability of each group was(105.298±9.395)%、(97.106±5.417)%、(77.015±4.055)%、(67.613±3.853)%and(43.872±9.532)%;However,the same concentrations(5,10,25,50,100μg/mL)of LDL treatment had no influence on cell viability(P>0.05),and the cell viability changes were(97.55±6.217)%,(99.640±3.586)%,(90.495±2.786)%,(83.552±9.171)%and(90.910±1.429)%respectively.Flow cytometry results showed that OxLDL with concentrations higher than 25μg/mL could induce apoptosis apparently.The apoptosis rates of the blank group,the OxLDL(25μg/mL)group,and the LDL(25μg/mL)group were(5.271±0.519)%,(41.23±1.686)%and(13.07±2.579)%respectively,and the differences among them were statistically significant(F=329.8,P<0.01);The Western blotting results showed that the expression levels of ERS-related proteins and apoptosis-related enzymes in the OxLDL(25μg/mL)group were significantly higher than those in the control group and the LDL group(Caspase-12:F=50.53,P<0.05;GRP78:F=55.60,P<0.05;CHOP:F=38.22,P<0.05;XBP-1:F=53.94,P<0.05;ATF6:F=20.01,P<0.05),while there was no difference between the control group and the LDL group(P>0.05).CONCLUSION:ERS is involved in the apoptosis of RPE cells induced by OxLDL,and regulating ERS may achieve the purpose of inhibiting RPE cell apoptosis and thus treating RPE apoptosis-related diseases.
作者
吴桐
党宽荣
苏静波
吕葆真
陆新婷
惠延年
杜红俊
Tong Wu;Kuan-Rong Dang;Jing-Bo Su;Bao-Zhen Lyu;Xin-Ting Lu;Yan-Nian Hui;Hong-Jun Du(Eye Institute of PLA,Department of Ophthalmology, Xijing Hospital, Air Force Medical University, Xi'an 710032, Shaanxi Province, China)
出处
《国际眼科杂志》
CAS
北大核心
2020年第10期1688-1692,共5页
International Eye Science
基金
国家自然科学基金资助项目(No.81470654)
陕西省自然科学基金资助项目(No.2019SF-047)。
关键词
年龄相关性黄斑变性
视网膜色素上皮
氧化应激
内质网应激
凋亡
age-related macular degeneration
retinal pigment epithelium
oxidative stress
endoplasmic reticulum stress
apoptosis
