Objective:To investigate the protective effect and molecular mechanism of human amniotic mesenchymal stem cell conditioned medium(h AMSC-CM)on retinal pigment epithelial cells(ARPE-19)damaged by oxidative stress,so as to provide theoretical basis for the search for a new method and new candidate for the treatment of retinal oxidative stress injury.Methods:1.Culture and identification of h AMSC:digestion method was used to isolate and culture h AMSC from amniotic membrane.Its morphology was observed and surface markers were detected by flow cytometry and immunofluorescence assay.2.Effect of h AMSC-CM on oxidative stress injury of ARPE-19 cells.The oxidative stress model of ARPE-19 cells was constructed by using H2O2.The effects of h AMSC-CM on the proliferation and apoptosis of damaged ARPE-19 cells were detected by CCK-8 and flow cytometry.3.Molecular mechanism of h AMSC-CM on oxidative stress injury of ARPE-19cells.(1)Fluorescence microscopy was used to detect the effects of h AMSC-CM on mitochondrial membrane potential MMP and reactive oxygen ROS levels of ARPE-19cells damaged by H2O2,laser confocal microscopy was used to detect mitochondrial morphological changes,and Western blot was used to detect apoptotic protein changes.To determine the effect of h AMSC-CM on mitochondrial apoptosis pathway in damaged ARPE-19 cells.(2)PI3K inhibitors LY294002 and Akt inhibitors TIC10 were added.Western Blot was used to detect the expression levels of PI3K/AKT signaling pathway related proteins in the normal group,H2O2 treated group,h AMSC-CM group and inhibitor group,and cell proliferation activity in each group was detected by CCK-8 method.Cell death in each group was detected by Calcein-AM/PI fluorescence double staining method,and mitochondrial function in each group was detected by fluorescence microscopy to determine whether h AMSC-CM affected mitochondrial apoptosis pathway of damaged ARPE cells through PI3K/AKT pathway.4.Effects of exosomes in h AMSC-CM on oxidative stress injury of ARPE-19 cells.Ultrafast centrifugal method was used to extract exosomes(h AMSC-exo)from h AMSC-CM.The exosomes were identified by electron microscopy and Western blot,and the effects of h AMSC-exo on the proliferative activity and apoptosis of H2O2-induced ARPE-19 cells were further detected.Results:1.h AMSC has mesenchymal stem cell characteristics.h AMSC was spindle-shaped and expressed embryonic stem cell markers OCT-4 and Nanog,mesenchymal stem cell markers CD90 and CD105,and did not express hematopoietic stem cell markers CD45 and CD34,major histocompatibility proteins HLA-DR and co-stimulators CD80 and CD40.2.h AMSC-CM protected ARPE-19 cells from H2O2-induced injury and inhibited their apoptosis.h AMSC-CM significantly increased the number of damaged APRE-19cells and decreased the number of apoptosis.3.h AMSC-CM regulates the mitochondrial apoptosis pathway through PI3K/AKT signaling pathway,thus affecting the proliferation and apoptosis of damaged ARPE-19 cells.h AMSC-CM effectively inhibited the decrease of mitochondrial membrane potential and ROS generation in damaged ARPE-19 cells,down-regulated the expressions of pro-apoptotic proteins Bax,Bim,Cleaved Caspase3,Cleaved Caspase9,up-regulated the expression of anti-apoptotic protein Bcl2,and affected mitochondrial morphology.4.h AMSC-CM may affect the PI3K/AKT pathway through exosomes and improve the proliferative activity of damaged ARPE-19 cells.h AMSC-exo was intact and spherical,expressed exosome marker proteins CD9 and CD81,and improved the activity of damaged ARPE-19 cells and reduced the apoptosis rate,while PI3K inhibitor LY294002 and Akt inhibitor TIC10 reversed the protective effect of h AMSC-exo on damaged APRE-19 cells.Conclusion:h AMSC-CM may regulate the PI3K/AKT signaling pathway through exosomes,affect the mitochondrial apoptosis pathway,and then improve the proliferation activity of damaged ARPE-19 cells and inhibit their apoptosis. |