抑制光损伤人视网膜色素上皮细胞血管内皮生长因子A的表达对趋化因子受体3配体表达的影响

Down-regulated expression of CC chemokine receptor 3 ligands in ranibizumab treated light-injured human retinal pigment epithelium cells

目的 观察抑制光损伤人视网膜色素上皮（RPE）细胞血管内皮生长因子A（VEGF-A）的表达对趋化因子受体3（CCR3）配体嗜酸细胞活化趋化因子（eotaxin）表达的影响.方法 体外培养人RPE细胞,取第8～12代生长良好的传代细胞用于实验.将同代细胞随机分为正常对照组、光照损伤组和光照干预组.采用波长400 nm的蓝色节能灯以（600±100） Lux持续光照RPE细胞12h建立光损伤模型,光照干预组采用0.125 mg/ml的VEGF-A受体拮抗剂雷珠单抗处理光损伤的RPE细胞;正常对照组以锡箔纸包裹细胞培养皿避光培养.结束光照24 h时,应用实时聚合酶链反应、酶联免疫吸附测定、蛋白免疫印迹法及免疫组织化学染色法检测3组细胞中VEGF-A、eotaxin-1、eotaxin-2、eotaxin-3及核因子（NF）-κB的mRNA和蛋白表达情况.结果 光照损伤组VEGF-A、eotaxin-1、eotaxin-2、eotaxin-3、NF-κB的mRNA和蛋白表达较正常对照组明显增加,差异均有统计学意义（P＜0.05）.光照干预组VEGF-A、eotaxin-1、eotaxin-2、eotaxin-3、NF-κB的mRNA和蛋白表达较光照损伤组明显降低,差异也有统计学意义（P＜0.05）.结论 抑制光损伤后人RPE细胞VEGF-A的表达可以下调CCR3配体eotaxin的表达,其机制可能与阻断转录因子NF-κB的激活有关.

Objective To observe the changes of eotaxin-1（CCL11）,eotaxin-2（CCL24）and eotaxin-3 （CCL26）in ranibizumab treated light-injured human retinal pigment epithelium （RPE） cells ARPE-19 and investigate the effects of vascular endothelial growth factor （VEGF） antagonist to the expressions of eotaxins.Methods Cultured human RPE cells （8th-12th generations）were divided into light-injured group,ranibizumab treated group and normal control group.Cells of the three groups were exposed to the blue light at the intensity of（600± 100） Lux for 12 h to establish the light injured model,while cell culture dishes of the normal control group were wrapped with double-layer foil.The cells of ranibizumab treated group were treated with VEGF-A antagonist（ranibizumab）at the final concentration of 0.125 mg/ml for 24 hours directly after the illumination.The mRNA and protein of VEGF-A,eotaxin-1,eotaxin-2,eotaxin-3,NF-κB were determined by Real time-PCR,enzyme-linked immunosorbent assay,Western blot,immunohistochemical staining at 0,3,6,12,24 hours after light damage.Results The mRNA and protein level of VEGF-A,eotaxin-1,eotaxin-2,eotaxin-3,NF-κB in the light-injuried group increased significantly compared to that in normal control group （P＜0.05）.After treating with ranibizumab,the expression of eotaxin-1,eotaxin-2,eotaxin-3,NF-κB were significantly suppressed （P ＜ 0.05）.Conclusion The suppression of over-expression of VEGF in human RPE may down-regulate the expression of eotaxins,via the suppression of NF-κB.