光照对人视网膜色素上皮细胞趋化因子受体3配体的影响

摘要

目的 观察光照后人视网膜色素上皮(RPE)细胞趋化因子受体3(CCR3)配体嗜酸性粒细胞趋化因子(eotaxin)-1、eotaxin-2、eotaxin-3的表达.方法 体外培养人RPE细胞,取第5～10代生长状态良好的传代细胞用于实验.将同代细胞随机分为光照干预组与正常对照组.采用15 W蓝色荧光灯自制光照器,以(600± 100) Lux的强度对光照干预组细胞持续光照12 h,对照组细胞采用双层高压消毒锡纸包裹.随后两组细胞均置于恒温培养箱内继续培养,并于光照结束后0、3、6、12、24 h终止细胞培养.采用实时聚合酶链反应和蛋白免疫印迹法分别检测eotaxin-1、eotaxin-2、eotaxin-3 mRNA和蛋白的表达.结果 光照干预组细胞eotaxin-1、eotaxin-2、eotaxin-3 mRNA和蛋白表达均随培养时间延长呈上升趋势,其中eotaxin-3 mRNA和蛋白表达增加最为明显.光照干预组与正常对照组比较,光照结束后0(t1=6.05,t2=12.561)、3(t1=2.95,t2=3.67)h时eotaxin-1、eotaxin-2 mRNA表达间差异均有统计学意义(P＜0.05),eotaxin-3 mRNA表达间差异无统计学意义(t3 =1.57、1.00,P＞0.05)；光照结束后6(t1 =4.73,t2=18.64,t3 =28.48)、12(t1=3.11,t2=20.62,t3=18.50)、24(t1=8.25,t2=38.27,t3=18.60)h时eotaxin-1、eotaxin-2、eotaxin-3 mRNA表达间差异均有统计学意义(P＜0.05).光照干预组与正常对照组比较,除光照结束后3h时eotaxin-3蛋白表达间差异无统计学意义外(t3=1.28,P＞0.05)；光照结束后0(t1=4.85,t2 =5.45,t3 =6.21)、3 h(t1 =5.64,t2=4.55)、6(t1 =31.60,t2 =6.63,t3 =7.15)、12(t1=14.09,t2 =18.22,t3=15.76)、24(t1 =6.96,t2 =10.47,t3=12.85)h时eotaxin-1、eotaxin-2、eotaxin-3蛋白表达间差异均有统计学意义(P＜0.05).结论 光照后人RPE细胞CCR3配体eotaxin-1、eotaxin-2、eotaxin-3 mRNA和蛋白表达均随培养时间延长呈上升趋势,以eotaxin-3表达增加最为突出.%Objective To investigate the expression of eotaxin-1,eotaxin-2 and eotaxin-3 in ARPE-19 human RPE cells after exposure to light.Methods Cultured human RPE cells (5th～ 10th generations)were divided into light-induced group and control group.Cells light-induced group were exposed to the blue light at the intensity of (600±100) Lux for 12 h to establish the light damaged model.Eotaxin-1,eotaxin2 and eotaxin-3 mRNA and protein were determined by real time polymerase chain reaction and Western blot at 0,3,6,12,24 hours after light-induced.Results In light-induced groups,mRNA levels of eotaxin-1and eotaxin-2 were increased at 0 h (t1 =6.05,t2 =12.561) and 3 h (t1 =2.95,t2 =3.67) significantly (P＜0.05),but the mRNA level of eotaxin-3 had not changed (t3 =1.57 and 1.00 respectively,P＞0.05)at that time.At6 h (t1=4.73,t2=18.64,t3=28.48),12 h (t1=3.11,t2=20.62,t3=18.50),24 h(t1 =8.25,t2=38.27,t3 =18.60),mRNA levels of eotaxin-1,2,3 were increased significantly (P＜0.05).Except for the eotaxin-3 protein had not changed at 3 h (t3 =1.28,P＞0.05),protein expression of eotaxin-1,2,3 were increased significantly (P＜0.05) at 0 h (t1 =4.85,t2 =5.45,t3 =6.21),3 h (t1 =5.64,t2 =4.55),6 h (t1 =31.60,t2 =6.63,t3 =7.15),12 h (t1 =14.09,t2 =18.22,t3 =15.76),24 h (t1 =6.96,t2 =10.47,t3 =1285).Conclusion Eotaxin-1,eotaxin-2 and eotaxin-3 expression were increased after light-damage,corresponding to the time after light exposure.Eotaxin-3 was the most prominent isoform.