Role Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosome On Degenerative Nucleus Pulpous Stem Cell

AIM:Exosomes are one of the new breakthroughs in stem cell research in recent years.More and more studies have shown that the main effect of implanted stem cells is not themselves,but the various substances they secrete,and exosomes are the main components of these substances.This study focuses on the role of umbilical cord mesenchymal stem cell-derived exosomes in degenerated intervertebral disc nucleus pulposus stem/progenitor cells(D-NPMSCs).The effect of exosomes is assessed primarily from cell proliferation,pluripotency and immune regulation.In addition,the mechanism is studied.Methods:Enzymatic digestion and explants culture were used to extract degenerated nucleus pulposus stem/progenitor cells and umbilical cord-derived mesenchymal stem cells(UC-MSCs)from degenerated nucleus pulposus tissue and healthy neonatal umbilical cord tissue.The cells were cultured to P3 and used for experiments.Exosomes were extracted from UC-MSCs conditioned medium by ultracentrifugation,and the extracted exosomes were identified by using transmission electron microscopy,nanoparticle analysis system and western blot.The obtained exosomes were applied to D-NPMSCs,and the expression of MSC-specific surface marker was detected by flow cytometry.The expression level of nucleus pulposus progenitor cell marker,inflammatory factors,pluripotent gene,nucleus pulposus cell specific gene and inflammatory factor was detected by qRT-PCR and western blot.The effects on proliferation,apoptosis and cell cycle of D-NPMSCs were detected by using CCK8 and flow cytometry.The protein expression of proliferation-related pathways was detected by western blot.Result:Identification of the vesicles extracted from the conditioned medium of UC-MSCs showed that 70% of the vesicles had a membrane-like structure with a particle size of 30-100 nm and a high expression of the exosome positive markers CD63 and TSG101.After D-NPMSCs were treated with the exosomes,the results showed that the expression levels of MSC-specific surface marker,nucleus pulposus stem/progenitor cells marker gene Tie2 and pluripotent gene SOX2,OCT4 and NANOG were significantly up-regulated,while NP cell specific marker ACAN,COL2A1 and CD24 were significantly down-regulated.The inflammatory factor IL-1? were significantly decreased,but the expression of IL-6 was significantly increased.Cell proliferation was significantly promoted,and cell apoptosis was decreased.Similarly,the expression of cell proliferation-related pathway protein was significantly increased.Conclusion:The experimental data indicated that exosomes secreted by human umbilical cord-derived mesenchymal stem cells can increase the cell proliferation and pluripotency gene expression of D-NPMSCs,and regulate the expression of inflammatory factors.Exosome enhanced the regenerative activity of D-NPMSCs.