人脐带间充质干细胞外泌体对蓝光损伤人视网膜色素上皮细胞血管内皮生长因子A表达的影响

Effect of exosomes derived from human umbilical cord mesenchymal stem cells on the expression of vascular endothelial growth factor A in blue light injured human retinal pigment epithelial cells

目的 观察人脐带间充质干细胞（hUCMSC）外泌体对蓝光损伤后人视网膜色素上皮（RPE）细胞血管内皮生长因子A（VEGF-A）表达的影响.方法 培养hUCMSC,收集上清液,采取梯度超速离心法分离纯化外泌体.应用透射电子显微镜观察外泌体形态;蛋白免疫印迹法（Western blot）检测hUCMSC外泌体表面特异性标志蛋白CD63及hUCMSC表面蛋白CD90的表达.取生长良好的人RPE细胞,随机分为正常对照组、光损伤组和hUCMSC外泌体处理组.光损伤组和hUCMSC外泌体处理组以蓝光（2000±500） Lux照射RPE细胞12h建立光损伤模型;hUCMSC外泌体处理组细胞培养液中分别加入25、50、75 μg/ml的hUCMSC外泌体,并以此分为低浓度、中浓度、高浓度3个亚组.各浓度亚组继续培养8、16、24 h结束培养.采用Western blot及免疫荧光检测各组RPE细胞VEGF-A蛋白表达,实时定量聚合酶链反应（RT-PCR）检测各组RPE细胞VEGF-A mRNA表达.结果 透射电子显微镜观察发现,hUCMSC外泌体为圆形或椭圆形膜性小囊泡,直径50～100 nm.Western blot检测结果显示,hUCMSC外泌体表达外泌体表面特异性标志蛋白CD63及hUCMSC表面蛋白CD90.Western blot及RT-PCR检测结果显示,光损伤组RPE细胞VEGF-A蛋白及mRNA表达较正常对照组明显增加,差异有统计学意义（t=-16.553、-19.456,P＜0.05）.hUCMSC外泌体处理组RPE细胞培养8、16、24 h时,低浓度、中浓度、高浓度亚组RPE细胞与光损伤组RPE细胞相比,VEGF-A蛋白及mRNA表达均有下降,差异均有统计学意义（P＜0.05）.随hUCMSC外泌体作用时间延长及作用浓度增强,RPE细胞VEGF-A蛋白及mRNA下调作用越强（P＜0.05）.免疫荧光检测结果显示,相同作用时间随着hUCMSC外泌体作用浓度提高,VEGF-A蛋白表达不断下降.结论 hUCMSC外泌体能够有效降低蓝光损伤后人RPE细胞VEGF-A的表达,其效应与hUCMSC外泌体作用浓度、时间呈正相关.

Objective To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells （hUCMSC） on the expression of vascular endothelial growth factor （VEGF） A in blue light injured human retinal pigment epithelial （RPE） cells.Methods hUCMSC were cultured with exofree fetal bovine serum for 48 hours,and then the supernatants were collected to isolate and purify exosomes by gradient ultracentrifugation method.Transmission electron microscopy was used to identify the morphology of exosomes.Surface specific maker protein CD63 and CD90 were detected via Western blot.Cultured ARPE-19 cells were divided into normal control group,blue light injured group and hUCMSC exosomes treated group.Cells were exposed to the blue light at the intensity of （2000 ± 500） Lux for 12 hours to establish the light injured models.The cells of hUCMSC exosomes treated group were treated by different concentrations of exosomes for 8,16,24 hours.The mRNA and protein of VEGF-A were determined by real time-polymerase chain reaction and Western blot.Immunofluorescence assay were used to detect the expression levels of VEGF-A.Results hUCMSC exosomes were successfully isolated,they exhibited round or oval shape and their diameter ranged from 50 to 100 nm with membrane structure through electron microscope,hUCMSC exosomes expressed the common surface marker protein CD63 and the surface marker protein CD90 of hUCMSC.The protein and mRNA level of VEGF A in the blue light injured group increased significantly compared to that in normal control group （t=-16.553,-19.456;P〈0.05）.After treating with low,middle and high concentration of hUCMSC exosomes for 8,16 and 24 hours,the protein and mRNA level of VEGF A of injured RPE were significantly decreased （P〈0.05）.With the treated time and concentration of hUCMSC exosomes improved,the protein and mRNA level of VEGF A of injured RPE gradually decreased （P〈0.05）.Immunofluorescence assay showed the protein level of VEGFA of injured RPE gradually decreased with the same concentration of hUCMSC exosomes treated over time.Conclusion hUCMSC exosomes can effectively down-regulate the mRNA and protein level of VEGF-A in blue light injured RPE,the effect depends on the concentration and treated time of hUCMSC exosomes.