Researches Of Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells On The Expression Of ICAM-1 And VEGF In Human Retinal Pigment Epithelial Cells Under High Glucose

Objective Observing the influence of exosomes of human umbilical cord blood mesenchymal stem cells(hUCMSCs)on the expression of intercellular adhesion molecule-1 of retinal pigment epithelium(RPE)cells and vascular endothelial growth factor(VEGF)after high sugar damage.Methods 1.Extraction and identification of exosomes of hUCMSCs.Culturing hUCMSCs and collecting the supernatant.And then separating and purifying the exosomes by gradient ultracentrifugation.The morphology of exosomes was observed by transmission electron microscopy.The expression of exosomes surface specific marker protein CD63 and the hUCMSCs surface protein CD90 was detected by Western Blot.2.Establishment of RPE cell injury model under high glucose.Human RPE cells(ARPE-19)with good growth were randomly divided into normal glucose concentration group: glucose concentration: 5.5 mmol/L,high glucose group I: 20.0 mmol/L;high glucose group II: 35.0 mmol/L: high glucose group III: 50.0 mmol/L;hypertonic control group IV: 5.5 mmol/L + mannitol 24.4 mmol/L;cultured ARPE respectively with different concentrations of glucose-19 cells and then tested the proliferation rate of ARPE-19 cells at different glucose concentrations by MTT assay.Western blot and immunofluorescence were used to detect the expression of ICAM-1 and VEGF protein in ARPE-19 cells at different glucose concentrations.The expressions of ICAM-1 and VEGF mRNA in ARPE-19 cells were detected by RT-PCR.After establishing the injury model,the RPE cells cultured under high glucose were treated with 25 ?g/ml,50 ?g/ml,75 ?g/ml of exosome of hUCMSCs,and the expression of RPE cells ICAM-1 and VEGFwere detected by Western Blot and immunofluorescence.Real-time polymerase chain reaction(RT-PCR)was used to detect the expression of ICAM-1 and VEGF-A mRNA in RPE cells.Results 1.Under the electron microscope,the exosomes of hUCMSCs were a group of tiny vesicles with membrane structures,whose diameter were 50-100 nm,and their cysts contained tiny electron vesicles with low electron density.The morphology was consistent with the related descriptions in the previous literature.In this study,western blot was used to detect the specific protein CD63 shared by exosomes and the surface specific protein CD90 of hUCMSCs,which confirmed that exosomes were derived from hUCMSCs.2.With the increase of glucose concentration,the cell proliferation rate gradually decreased,and the expression of ICAM-1,VEGF protein and mRNA increased.The differences were statistically significant(P<0.05).There was no significant difference between the expression of ICAM-1 and VEGF protein and mRNA in the hyperosmotic group and the normal glucose concentration group.There was no significant difference between the two groups(P>0.05).After the culture of RPE cells in different concentrations of hUCMSC exosome for 24 h,the expression of ICAM-1,VEGF protein and mRNA decreased compared with the high glucose group.The differences were all statistically significant(P < 0.05).Moreover,the down-regulation of ICAM-1 by hUCMSC exosomes increased with the concentration of exosome in hUCMSC(P<0.05).Conclusion 1.The supernatant of hUCMSCs can be used to extract stable dose exosomes by gradient ultracentrifugation.The surface specific antigen detection verified the reliability of its source.2.In the 35.0 mmol/L glucose concentration group,cells were able to express ICAM-1 and VEGF higher,but the apoptotic rate was not high.Therefore,this study selected a medium concentration of glucose: 35.0 mmol / L as the experimental cell damage model.3.The exosomes of hUCMSCs were able to effectively reduce the expression of ICAM-1 and VEGF in ARPE-19 cells under high glucose,and positively correlated with the concentration of action.