The Effect Of Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes On Immunoregulation And Autophagy Level Of Macrophage

ObjectivesTo detect the role of the human umbilical cord mesenchymal stem cells derived exosomes(hucMSC-EXO)in regulating the immune function and autophage function of macrophage,by coculturing the hucMSC-EXO with macrophage.Methods1.Using the ultracentrifugation and ultrafiltration method to isolate and purify the hucMSC-EXO.(1)Marker proteins of hucMSC-EXO,such as CD9、CD81、Tsg101、Calnexin were detected by Western blot.(2)The size of hucMSC-EXO was dected by Nanoparticle Tracking Analysis method.(3)The morphology of hucMSC-EXO was identified by transmission electron microscope.2.PKH-67 labeled hucMSC-EXO was cocultured with macropahe for 1 hour,2 hours,3hours,4 hours,8 hours,24 hours 48 hours and 72 hours.Fluorescence confocal microscope was used to examine the uptake of PKH-67 labeled hucMSC-EXO by macrophage.3.hucMSC-EXO was cocultured by macrophage stumilated by LPS and IFN-γ for 24 hours,the genes expression level of TNF-α、IL-10、IL-1β were detected using the RT-PCR.The concentration of IL-1β,IL-6,IL-10 in the cell culture supernatant was detected by ELISA method.4.After macrophage cocultured with hucMSC-EXO for 24 hours,Western blot method was used to detect the protein expressions of autophagy-related proteins such as p-AMPK,p-AKT,p-m TOR,p-ULK1,Beclin 1,ATG 7,LC 3 and etc in macropahge.Results1.Western blot results showed that the isolated hucMSC-EXO could express the protein CD9,CD81,TSG101,but Calnexin.The diameter of the hucMSC-EXO was mainly about131.9 nm using the Nanoparticle Tracking Analysis method.Under the transmission electron microscrope,the hucMSC-EXO was the cup shaped vesicle.All the results indicated the hucMSC-EXO could be extracted from the hucMSC culture medium.2.Using the fluorescence confocal microscope,the intensity of PKH-67 labeled hucMSC-EXO in macrophages gradually increased from 1 hour,2 hours,3 hours,4 hours,8 hours to 24 hours,and reached the peak at about 24 hours,and decreased at 48 hours and 72 hours.3.RT-q PCR revealed that hucMSC-EXO downreagulated the expressions of TNF-α in macrophage stumilated by LPS and IFN-γ.And the ELISA results revealed hucMSC-EXO downregulated the the expressions of IL-1β,IL-6 but upregulated the expressions of IL-10 in macrophage stumilated by LPS and IFN-γ.4.Western blot method revealed that the expressions of p-AMPK,p-AKT,p-m TOR,Beclin1,ATG 7,LC3-Ⅱ expression were significantly higher in macrophage cocultured with hucMSC-EXO than in control,while the protein p62 and p-ULK1 were lower than in control.Conclusions1.The hucMSC-EXO could promote the release of anti-inflamation cytokines,but inhibited the inflammatory cytokines in macrophage.2.The hucMSC-EXO activated the AMPK and AKT pathway and autophage in macrophage.