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。

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, 100 μg/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, 100 μg/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.