Researches Of Exosomes Derived From Mesenchymal Stem Cells On The Expression Of Vascular Endothelial Growth Factor-A In Blue Light Injured Human Retinal Pigment Epithelial Cells And Laser Induced Choroidal Neo-vascularization In Rats

Objective To observe Effect of exosomes derived from human umbilical cord blood mesenchymal stem cells on the expression of VEGF A in which blue light injured human retinal pigment epithelial cells and laser induced choroidal neovascularization in rats.Methods 1.The isolation and identification of h UCMSCs exosomes.Human umbilical cord blood mesenchymal stem cells were cultured with exo-free FBS for 48 h,then we collected the the supernatant to isolate and purify exosomes by using 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.2.Effect of h UCMSCs exosomes on the expression of VEGF A of blue light injured human retinal pigment epithelial cells.Cultured ARPE-19 cells were divided into normal control group,blue light injured group and h UCMSCs exosomes treated group.Cells were exposed to the blue light at the indensity of(2000士500)Lux for 12 h to make the light injured models.The cells of h UCMSCs exosomes treated group were treated by different concentrations of exosomes for 8,16,24 hours.The m RNA and protein of VEGF-A were determined by Real time-PCR and western blot.Immunofluorescence assay were used to detect the expression levels of VEGF-A.3.Effect of h UCMSCs exosomes on the expression of VEGF A of laser induced choroidal neovascularization in rats.Totally 90 BN rats were randomly divided into 6 groups: normal control group,model group,low,medium and high dose h UCMSC exosomes treated group,physiological saline group,15 rats in each groups.All groups of BN rats except normal control group were treated with krypton red laser,the parameters of laser are: 577 nm,200mw,100μm,0.1s.1μl,2μl,3μl h UCMSCs exosomes and 2μl physiological saline were respectively injected in vitreous body immediately after photocoagulation according to the groups.Leakage of choroidal of rats of each groups on the 14 th day and the 21 st day after laser treated via fundus fluorescein angiography.Histological structure of CNV of rats were inspected by HE staining.The expression of VEGF-A was detected withimmunohistochemical method.Results 1.HUCMSC exosomes were successfully isolated,they exhibited orbiculate shape and their diameter ranged from 50 to 100 nm with membrane structure through electron microscope.HUCMSCs exosomes expressed the common surface marker protein CD63 and the surface marker protein CD90 of h UCMSCs.2.The protein and m RNA level of VEGF A in the blue light injuried group increased significantly compared to that in normal control group(P<0.05).After treating with low,middle and high concentration of h UCMSCs exosomes for 8,16 and 24 hours,the protein and m RNA level of VEGF A of injured RPE were significantly decreased(P<0.05).With the treated time and concentration of h UCMSCs exosomes improved,the protein and m RNA level of VEGF A of injured RPE gradually decreased(P<0.05).Immunofluorescence assay showed the protein level of VEGF-A of injured RPE gradually decrease with the same concentration of h UCMSCs exosomes treated over time.3.The leakage of laser point in different dose h UCMSCs exosomes treated groups lightened significantly compared to that in model group(P<0.05).With the dose of h UCMSCs exosomes improved,the leakage of laser point gradually decreased(P<0.05).The protein level of VEGF-A of laser points of rats of h UCMSCs exosomes treated groups decreased significantly compared to that in model group(P<0.05).With the dose of h UCMSCs exosomes improved,the protein level of VEGF-A of laser points gradually decreased(P<0.05).Histological structures of CNV are gradually improved with the increased h UCMSCs exosomes treated.Conclusion 1.Exosomes can be extracted from the h UCMSCs supernate by density gradient ultracentrifugation.Properties of h UCMSCs exosomes are certified by the test of CD63 and CD90 on their surfaces.2.HUCMSCs exosomes can effectively down-regulate the m RNA and protein level of VEGF-A in blue light injured RPE,the effects depends on the concentration and treated time of h UCMSCs exosomes.3.HUCMSCs exosomes can effectively lighten the leakage of laser point of rats and down-regulate the protein level of VEGF-A of laser induced rats,the effects depends on the dose of h UCMSCs exosomes.