蓝光对人视网膜色素上皮细胞线粒体膜电位及细胞色素C的影响

The Effect of Blue Light on Mitochondrial Membrane Potential and Cytochrome C of Cultured Human Retinal Pigment Epithelium Cells

目的 探讨蓝光导致人视网膜色素上皮 (retinal pigm ent epithelium,RPE)细胞损伤的分子机制 ,明确在此过程中线粒体膜电位及细胞色素 C(cytochrom e C,Cyt C)是否发生变化。方法 用蓝光照射体外培养的人 RPE细胞 ,罗丹明 12 3荧光染料孵育人 RPE细胞 ,流式细胞仪检测线粒体膜电位 (ΔΨm)。分为 3组 ,A组 :不同光照强度 ;B组 :同一光照强度 ,不同光照时间 ;C组 :同一光照强度和光照时间 ,不同细胞培养终止时间。用酶联免疫法测定Cyt C的活性 ;比色测定 Caspase- 3的活性。分为两组 :(2 0 0 0± 5 0 0 ) lx,光照 6 h为 组 ,光照 12 h为 组。结果 A组线粒体膜电位随着光照强度增加 ,呈逐渐下降趋势 ;B组在光照 3h,RPE细胞荧光强度无明显变化 ,当光照 6 h,荧光强度明显下降 ;C组光照后 6 h,荧光强度开始明显下降 ,一直持续到光照后 4 8h。 组和 组光照后 2 4及 36 h,Cyt C的浓度明显升高 ,光照后 2 4 h,后者 Cyt C浓度的升高值明显高于前者。未检测到 Caspase- 3活性变化。结论 蓝光照射导致培养的人 RPE细胞损伤过程与线粒体膜电位降低、细胞色素

Objective To answer the question whether mitochondrial permeability transition (MPT) participates in blue light-induced damage to human retinal pigment epithelium (RPE) cells, this study was directed at assessing the effect of blue light on mitochondrial membrane potential (ΔΨ m) and cytochrome C (Cyt C) of cultured human RPE cells. Methods Human RPE cells were exposed to blue light (wave length 470-490 nm); ΔΨ m was measured by rhodamine 123 staining and subsequent flow cytometry. Three groups were investigated: Group A (exposure to different intensity of blue light); group B(exposure to identical intensity for different duration); group C(exposure to identical intensity and duration, different prolongation of post-exposure culture). Cyt C activity was assayed by ELISA. Caspase-3 was detected by colorimetric assay. In these aspects, two groups were investigated: Group Ⅰ 〔(2000±500) lx for 6 h〕; Group Ⅱ 〔(2000±500) lx for 12 h〕. Results When human RPE cells were exposed to blue light, the more pronounced decrease of ΔΨ m was consistent with the increase of light intensity in group A. Pronounced decrease of ΔΨ m was seen at 6 h and 12 h of exposure duration in group B. At 6 h prolongation of post-exposure culture in group C, the decrease of ΔΨ m was observed, lasting 48 h. The concentration of Cyt C was detected; no significant changes were found at 6 h and 12 h prolongation of post-exposure culture, but a significant increase was found at 24 h and 36 h post-exposure in the two groups. The increase was more significant in Group Ⅱ than in Group Ⅰ at 24 h post-exposure. The activity change of caspase-3 was not found in the two groups. Conclusion Blue light exposure over threshold can induce damage to human RPE cells, probably by triggering the mitochondrial permeability transition, which results in the decrease of ΔΨ m and the release of cytochrome C.