Objective To research the role of mitochondrial DNA mediate the cultured human retinal pigment epithelium (hRPE) cell apoptosis induced by blue light and the relationship with time.Methods Established the blue light damage model of cultured hRPE cells in vitro with light emitting diode (LED) blue light density of (4.0-±0.5)mW/cm2 adjusted by FL-1D blue light illumination meter,and the illumination time was set as 0,0.5,1,2,4,6,12 and 24 hours,then the cells were grouped according to the illumination time.Immunofluorescence were used to identify the cells;the expressions of caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,bax and bcl-2 were detected with Western blot.Quantitative PCR was used to detect the copy number of mitochondrial DNA and PCR was used to detect mitochondrial DNA 4977bp common deletion.Results Immunofluorescence results showed that the RPE65 protein was expressed in the cytoplasm.The expressions of bax were upregulated after illumination for 1 hour,cleaved caspase-3 were upregulated after illumination for 2 hours,caspase-3,caspase-9,cleaved caspase-9 were upregulated after illumination for 4 hours,while the expression of bcl-2 was downregulated after illuminated for 2 hours,with significant differences compared to the normal control group (all at P＜0.05).The copy number of mitochondrial DNA in 0.5,1,2,4,6,12 and 24 hours groups was downregulated,with significant differences compared to the normal control group (all at P＜0.05).The expressions of 4977bp common deletion in 0.5,1,2,4,6,12 and 24 hours groups were increased,with significant differences compared with the normal control group (all at P＜0.05).Conclusions Blue light can cause cell apoptosis,especially mitochondrial apoptosis,in hRPE probably motivated by mitochondrial DNA damage.%目的 研究线粒体DNA参与蓝光诱导体外培养的人视网膜色素上皮(hRPE)细胞凋亡的作用机制及与时间的关系.方法 用发光二极管(LED)蓝光光源造成蓝光损伤体外培养的hRPE细胞模型,蓝光辐照度为(4.0±0.5)mW/cm2,并将细胞分为光照0(正常对照组)、0.5、l、2、4、6、12和24 h处理组.免疫荧光染色鉴定人原代hRPE细胞;Western blot法检测caspase-3、cleaved caspase-3、caspase-9、cleaved caspase-9、bax和bcl-2蛋白的表达情况;实时荧光PCR检测线粒体DNA拷贝数;普通PCR检测线粒体DNA 4977bp缺失.结果 分离的细胞呈长梭形、三角形或不规则形,RPE65特异地表达在细胞质中.凋亡蛋白bax从光照后1h显著上调;cleaved caspase-3从光照后2h开始表达水平均显著高于正常对照组;caspase-3、caspase-9和cleavedcaspase-9从光照后4h开始表达水平均显著高于正常对照组;bcl-2从光照后2h开始表达水平均较正常对照组下调,与正常对照组比较凋亡相关蛋白的表达差异均有统计学意义(均P＜0.05).实时荧光PCR检测正常对照组线粒体DNA拷贝数较光照0.5、1、2、4、6、12和24 h处理组降低,差异均有统计学意义(均P＜0.05).普通PCR检测光照0.5、1、2、4、6、12和24 h处理组线粒体DNA 4977bp缺失表达水平高于正常对照组,差异均有统计学意义(均P＜0.05).结论 蓝光可导致线粒体DNA损伤,进而激活细胞凋亡系统,尤其是线粒体凋亡通路,导致细胞损伤.
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