氧化应激诱导视网膜色素上皮细胞中钙蛋白酶激活作用研究

Calpain activation in retinal pigment epithelium( RPE) cells induced by oxidative stress

目的观察一定浓度H_2O_2氧化应激体外培养人视网膜色素上皮(retinal pigment epithelium,RPE)细胞中钙蛋白酶(Calpain)含量及细胞活性改变。方法常规培养商品化人RPE细胞系ARPE-19作为对照组,培养基内加入终浓度为10μmol·L^(-1)及100μmol·L^(-1)的H_2O_2作为实验组,培养4 h、8 h、16 h及32 h进行测定。MTT法测定不同浓度H_2O_2刺激下RPE细胞不同检测时间点细胞增殖率的变化;Fluo-3/AM、ester钙离子荧光探针标记细胞内钙离子,激光共聚焦显微镜及流式细胞仪检测RPE细胞内钙离子荧光值,间接观察钙离子浓度变化。10μmol·L^(-1)H_2O_2氧化刺激RPE细胞8 h,用WesternBlot法及实时荧光定量PCR法检测细胞内Calpain蛋白及RNA含量的变化。结果与对照组相比,10μmol·L^(-1)H_2O_2刺激RPE细胞4 h、8 h、16 h及100μmol·L^(-1)H_2O_2刺激RPE细胞4 h及8 h后增殖率(r值)升高差异均具有显著统计学意义(均为P<0.01)。100μmol·L^(-1)H_2O_2刺激16 h及32 h细胞增殖率均较对照组下降(均为P<0.01)。10μmol·L^(-1)H_2O_2刺激RPE细胞8 h后胞内钙离子荧光强度(563.27±77.10)U较对照组(170.77±20.11)U显著升高(P<0.01)、Calpain在m RNA和蛋白水平均较对照组显著升高(均为P<0.05),而在同时使用Calpain抑制剂SNJ-1945(SNJ)的实验组这些变化可被有效抑制。结论 RPE细胞中钙超载激活Calpain参与H_2O_2氧化刺激RPE细胞的氧化应激改变。

ObjectiveTo investigate the content of Calpain in H2O2 induced oxidative stress in cultured human retinal pigment epithelium(RPE)and the change of biologic activity of RPE cells.MethodsNormal culture of human RPE cell line(ARPE 19 cells)was assigned as the control group,while cells were cultured in the medium with 10μmol·L-1 and 100μmol·L-1 H2O2 were the experimental groups.After culture of 4 h,8 h,16 h,32 h,the proliferation rate of RPE cells induced with different concentrations of H2O2 was measured by methyl thiazolyl tetrazolium(MTT)assay,and the Ca2+concentration was measured with fluorescence dye Fluo 3/AM ester,laser scanning confocal microscopy(LSCM)and flow cytometry.Then irritation with 10μmol·L-1 H2O2 for 8 hours,Calpain protein and mRNA in RPE cells were detected by Western Blot and RT PCR.ResultsThe proliferation rates(r)of the RPE cells were increased significantly induced by 10μmol·L-1 H2O2 for 4 h,8 h,16 h and 100μmol·L-1 H2O2 for 4 h,8 h when compared with the control group,and the differences were statistically significant(all P<0.01),whereas the rates of the cells induced by 100μmol·L-1 H2O2 for 16 h and 32 h were less than those of the control group(all P<0 01).The Ca2+concentration in RPE cells induced by 10μmol·L-1 H2O2 for 8 h was significantly higher than that in the controls[(563.27±77.10)U vs.(170.77±20.11)U](P<0.01),and the protein and mRNA levels of Calpain were significantly increased when compared with the control group(both P<0.05).However,these changes can be effectively suppressed using Calpain inhibitor SNJ 1945(SNJ).ConclusionThe Ca2+inflow can make Calpain activation involve in oxidative stress stimulated by a certain concentration of H2O2 in RPE cells.