Influence Of Retinal Pigment Epithelium Cells Exosomes Affected By Oxidative Stress On Akt And VEGF-A Of Retinal Pigment Epithelium Cells

Objectives To investigate the effects of cultured human retinal pigment epithelium(ARPE-19) cells exosomes affected by oxidative stress on the proliferation of ARPE-19 cells, its impact on the changes of VEGF-A 、 Akt, then provide experimental evidences for the pathogenesis of AMD.Methods 1. culture human retinal pigment epithelium( ARPE-19) cells,supernatants were collected for exosomes purification. Exosomes were isolated by three methods: multi-step differential centrifugation、Exo Quick exosome precipitation solution、q EV size exclusion columns.Transmission electron microscopy(TEM) was used to identify the morphology of exosomes.Western blot was used to detect exosomes’ surface-specific maker protein CD63.Cell culture medium was added at a concentration of 0,1,2.5,5 μ mol/L rotenone,and analyzed the effect of different concentrations of rotenone on the proliferation of ARPE-19 cells.2. The concentration of 2.5μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome.Normal ARPE-19 cells were cultured with exosome.The cell proliferation was examined by MTT assay.Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein.RT-PCR was used to detect the levels of VEGF-A m RNA and Akt mRNA.Results 1. The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm.The isolated exosomes expressesd CD63.Rotenone can inhibit the proliferation of ARPE-19 cells.The inhibitory effect increased with the increase of concentration of rotenone.The difference was statically significant(p<0.05).2. AREP-19 cells were cultured with ARPE-19(affected by rotenone)-exosome,the cell viability in experimential grop was significantly reduced than in the control group. The difference was statically significant(p=0.002).Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the contral group,VEGF-A was up-regulated expressed and Akt was down-regulated expressed.Western blot results showed that,VEGF-A protein expression in the experimental group were higher than the control group(p=0.001).Akt protein expression in the experimental group were less than the control group(p=0.003). The difference was statically significant.RT-PCR results showed that VEGF-A m RNA espression levels was higher in the experimental group than the control group(2.37±0.43 vs 0.54±0.07).Akt m RNA espression levels was lower in the experimental group than the control group(0.64±0.11 vs 3.05±0.58). The difference was statically significant(p<0.05).Conclusion ARPE-19 cells can secrete exosomes having biological functions. ARPE-19 cells affected by oxidative stress exosomes inhibit the proliferation of normal ARPE-19 cells,increase the expression of VEGF-A and reduce the expression of Akt.