Preliminary Study On The Repair Of Human Endometrial Injury By Exosomes Derived From Umbilical Cord Mesenchymal Stem Cells

ObjectiveTo observe the recovery potential of exosomes derived from human umbilical cord mesenchymal stem cell(h UCMSC)for the treatment of damaged human endometrial cells in vitro.Methods1.Exosomes were extracted from human umbilical cord mesenchymal stem cells.The specific antibodies of human umbilical cord mesenchymal stem cells were detected by flow cytometry.The differentiation ability of stem cells was identified by multiple induced differentiation experiments.The morphology and size of exosomes from h UCMSC-Exos were observed by electron microscopy and Flow nano analyzer.Western blot was used to detect the expression of exosome markers.2.Endometrial stromal cells(HEndo SCs)were extracted from human endometrial tissue.The immunophenotype of HEndo SCs was detected by flow cytometry and the immunohistochemical identification showed the express of vimentin and keratin.3.The damaged endometrial stromal cell model was established by mifepristone in vitro.4.Cocultured mifepristone injured human HEndo SCs with h UCMSC-EXOs.Confocal microscopy detection of PKH26 labelled h UCMSC-Exos uptake by h Endo SCs at different time.The growth and apoptosis of mifepristone injured HEndo SCs were measured by flow cytometry and cck8.Meanwhile,we compared the activation of anti-apoptotic factors and AKT signaling pathway by western blot.Result1.The positive markers,including CD73,CD90 and CD105,were highly expressed in h UCMSCs.The negative markers CD34,CD45 and HLA-DR were not expressed.The differentiation potential of h UCMSCs into adipocytes,osteoblasts,chondroblasts was confirmed Western blot confirmed that h UCMSC-Exos expressed exosomal specific markers CD63,PDCD6 IP and TSG101,but not the negative marker autophagosome proteins LC3 A.Flow nano analyzer analysis showed that the average diameter is 86.79±22.70 nm and the concentration of the h UCMSC-Exos was approximately 3.17×1011 particles/m L.2.The immunophenotypes of h Endo SCs showed the cells were positive for CD34,CD44,and CD90.and the immunohistochemical identification showed that the vimentin and keratin were expressed in h Endo SCs.3.The confocal scanning laser microscopy showed the exosome uptake efficiency increased with the number of h UCMSC-Exos adsorbed or engulfed by the injured h Endo SCs.4.CCK8 and Flow cytometry showed h UCMSC-Exos promote the proliferation of damaged h Endo SCs,and protect h Endo SCs from mifepristone-induced apoptosis in vitro5.The expression of Caspase-3 and Cleaved Caspase-3 in mifepristone injured h Endo SCs was reduced as h UCMSC-Exos addition,and the Bcl-2 expression was increased.Exosome activates the PTEN/AKT signaling pathway,which regulates proliferation and inhibits apoptosis.ConlusionExosomes from human umbilical cord mesenchymal stem cells express correct markers,they can be can be ingested by mifepristone injured endometrial stromal cells.We found h UCMSC-Exos improved the proliferation of damaged h Endo SCs and protected h Endo SCs from the mifepristone induced apoptosis.h UCMSC-Exos upregulated Bcl-2 level as well as down-regulated Cleaved Caspase-3 level,and activated PTEN/AKT signaling pathway to regulate the proliferation and anti-apoptosis.In the clinical treatment group,the endometrium of the patients increased to different degrees,including 4 pregnancies,1 biochemical pregnancy and 1 embryo stop.