Effects Of HEMSC,hADSC And HUCMSC Extracellular Matrix On Fibroblasts Injured By H₂O₂

Purpose1.Exosomes were obtained from hEMSC,hADSC and hUCMSC conditional media,identified and compared;2.Establish an appropriate oxidative stress model with H₂O₂induced human primary fibroblasts;3.The objective of this study was to investigate the effects of hEMSC,hADSC,hUCMSc and their conditional medium on oxidative stress injured fibroblasts.Method1.hEMSCs were obtained from endometrial tissues with enzymatic isolation method and cultured for identification.hADSC and hUCMSC were cultured and amplified,and then we collect their conditioned media.hEMSC-derived exosomes were isolated by exosome kit and ultracentrifugation for comparision,and hADSC and hUCMSC mesenchymal stem cell-derived exosomes were isolated by ultracentrifugation only.The exosomes were characterized through transmission electron microscopy（TEM）,nanoparticle tracking analysis（NTA）。The quantity of protein content of exosomes was compared by BCA determination of protein concentration.2.In order to establish an appropriate oxidative stress model,the appropriate concentration of DCFH-DA was firstly determined,and the ROS fluorescence intensity of fibroblasts treated by different concentrations of H₂O₂solution was measured by flow cytometry。The stimulated fibroblasts were stained with live/dead cells to determine the appropriate concentration of H₂O₂solution。3.The appropriate oxidative stress model was determined by stimulating the fibroblasts with 100μM H₂O₂solution for 1 hour。The uninjured or injured fibroblasts were incubated with FBS-free DMEM basal medium、hEMSC、hADSC and hUCMSC conditional medium.After 24 hour,the ROS fluorescence intensity of fibroblasst was measured by flow cytometry。Results1.The exosomes derived from hEMSC、hADSC and hUCMSC were successfully isolated。The size of the exosomes measured by NTA was between 100 and 150nm,and TEM showed that the exosomes presented a double-membrane vesicle structure with typical saucer-like appearance。The concentration of BCA protein showed hEMSC-derived exosome protein（P<0.05）has the highest concentration,The concentration of exosome protein derived from hUCMSC was the lowest（P<0.05）。2.The concentration of 8μM DCFH-DA probe was determined as the probe in the experiment。The oxidative stress model of fibroblasts was stimulated with 100μM H₂O₂solution for 1 hour,which was used as the research model for the experiment。3.The experimental results showed that the ROS level of fibroblasts after 1 hour of stimulation with 100μM H₂O₂was higher than that of blank control group。After 24hours of incubation with basal medium or conditioned medium,incubation with FBS-free DMEM medium decreased ROS fluorescence intensity,incubation with hEMSC、hADSC and hUCMSC conditional medium increased ROS level of damaged fibroblasts,and hUCMSC conditional medium was the most damaged（P<0.05）。ConclusionThe ROS fluorescence intensity of the fibroblasts induced with H₂O₂was significantly increased in the conditioned media of hEMSC、hADSC and hUCMSC,and this phenomenon indicate that the conditioned media of hEMSC、hADSC and hUCMSC aggravated the oxidative stress induced injury of fibroblasts。...