Study On The Role And Mechanism Of Human Nucleus Pulposus Exosomes In Differentiation Of Human Urine-derived Stem Cells Into Nucleus Pulposus Cells

ObjectiveTo investigate the role of exosomes derived from human nucleus pulposus cells(NPCs)in inducing differentiation of human urine-derived stem cells(USCs)into nucleus pulposus cells,and to investigate the biological effects of these exosomes.MethodsMorning urine was collected from healthy volunteers and human urine-derived stem cells were extracted and cultured by centrifugation.The surface markers and differentiation of urine-derived stem cells were evaluated by flow cytometry and three-line induced differentiation.Protruding nucleus pulposus tissue was obtained through lumbar foraminoscopy,and NPCs were extracted after digestion.NPCs exosomes were extracted from supernatant by supercentrifugation,and surface markers were detected by transmission electron microscopy,particle size analysis and western blot.PKH26 dye-labeled NPCs exosomes were co-incubated with P3 USCs transfected with GFP lentivirus for 12 h,and the uptake of NPCs exosomes by USCs was observed under microscope.The experiment was divided into three groups: exosomes group,USCs and NPCs exosomes were co-cultured;In the co-culture group,USCs and NPCs Transwell cells were cultured.Control group,USCs culture alone.Fluorescence quantitative PCR(QRT-PCR)was used to detect the m RNA relative expression levels of nucleus pulposus marker genes in the three groups of USCs,and the induction effects were quantitatively compared.Cck-8 method was used to detect the effects of the three treatments on USCs proliferation.ResultsThe osteoblast,lipogenic and chondrogenic stem cells were successfully differentiated,and flow cytometry analysis showed high expression of CD29(99.57%),CD44(97.46%)and CD73(97.71%),low expression of CD31(0.59%)and CD45(0.19%).The exosomes extracted from the nucleus pulposa cells showed an oblate double-membrane structure of 50-100 nm by transmission electron microscopy,and the expression of CD63,CD81 and Tsg101 proteins was positive by western blot,but the negative protein Calnexin was not expressed.Fluorescence microscope observation showed that NPCs exosomes labeled with PKH26 red dye appeared in the cytoplasm of USCs transfected with GFP green fluorescence,which confirmed that USCs can take NPCs exosomes in vitro.Cck-8 cell proliferation experiment showed that USCs cell proliferation in exosome group was significantly higher than that in co-culture group and control group,with statistical significance(P < 0.05).Qrt-pcr showed that m RNA expression levels of nuclear nucleus pulposus marker genes(including COL2A1,ACAN,HIF-1α and SOX-9)in exosome group were significantly higher than those in control group and co-culture group,with statistical significance(P < 0.05).ConclusionThis study demonstrated that the nucleus pulposus exosomes can promote the directed differentiation of human urine-derived stem cells into nucleus pulposus like cells.Exosome induction has obvious inducing effect and cell proliferation.