Roles And Mechanism Of Exosome Derived From Human Umbilical Cord Mesenchymal Stem Cells In Spinal Cord Injury Repair

Objective:Spinal cord is one of the most important components of the central nervous system.Spinal cord injury usually results from traffic accidents,falls and sports injuries that could lead to severe sensory abnormalities and physical motor dysfunction blow the injury level which brings a heavy psychological and economic burden to patients and their families.Unfortunately,effective method is lack to cure this condition.Reconnection of electrical signal pathway becomes vital importance of repairing the SCI.Currently,stem cell-based replacement therapy has been widely reported and has made great progress.Exosome derived from stem cells was safer compare to the stem cell-based replacement therapy by avoiding the risk of tumor formation and immune rejection.The main purpose of this present study was to investgate the role and molecular mechanism of exosomes derived from human umbilical cord mesenchymal stem cells in spinal cord transection injury in SD rat models.Method:Extraction of hucMSC-Exs from condition medium of hucMSCs,and identified by western blot,nanosight and TEM.The cellular markers,diameter and cup-shape structure of exosomes were detected,respectively.Function of the spinal cord was analyzed by BBB score.Fluoscent-gold retrograde stain was ultilized to reflect the recirculation strace of tranverse spinal cord and staining of NF-200,Gap-43,SYP and PSD95 represents the neurongenesis in the original site of trauma.In order to further explore and explain the causes and repairing methods of hucMSC-Exs repairing spinal cord transection injury.we explore the possible roles and pathways of hucMSC-Exs in spinal cord transection injury from the following aspects.Degradation of the spinal cord and cavity were calculated by H&E stain of the spinal cord.Inflammatory factors of TNF-αand IL-1βwere detected by ELISA.Neural stem cell markers Nestin and Pax-6 were chosen to reflect the distribution of NSCs in the lesion site.In our vitro experiments,β-Ⅲ-tubulin staining of DRGs represents the roles of hucMSC-Exs on the outgrowth of axonal fibers,and then migration of NSCs was analyzed by transwell and radial migration assays.To investgate the promotion of hucMSC-Exs on the proliferation and differentiation of NSCs,cell viability and poliferative markers PCNA,Bcl-2 and Ki-67 were analyzed.Staining and Western blot for neuron and synapse molecules to indicate the differentiation of NSCs.ERK1/2 pathway inhibitor was utilized to explore the roles of hucMSC-Exs in promoting NSCs to proliferation and differentiation.Results:Western blot results indicated that exosome surface markers CD9 and CD63 were positive in hucMSC-Exs and negative in hucMSCs.Transmission electron microscopy revealed a characteristic cup-like structure of exosomes and median size of exosome was 71.50 ± 20.08 nm detected by nanosight.H&E staining shown that glial scars and carvity appeared in the original lesion site of spinal cord and were inhibited in hucMSC-Exs treated rat models,the degradation site is much larger in the injury rat models.BBB score shown that hucMSC-Exs could enhance the functional recovery of the traumatic spinal cord.Meanwhile,fluoscent-gold retrograde stain illustrated that hucMSC-Exs promote the the reconstruction of the eletrical pathway conduction through the lesion site.All above,our results demonstrated that hucMSC-Exs could enhance the functional recovery and repair of transverse spinal cord injury.Staining of the spinal cord found that the expression level of the mature neuron marker NF-200 and regenerated neuron marker Gap-43 were higher in the hucMSC-Exs treated rat models.Then the sypase markers SYP and PSD95 were also upregulated in the hucMSC-Exs treated rat models.HucMSC-Exs enhance regeneration of neurons and synapse.ELISA assay results illustrated that expression level of the TNF-βand IL-1αdecreased which indicates that hucMSC-Exs could attenuate inflammation in SCI rats.HucMSC-Exs could enhance the proliferation and nural differentiation of neural stem cells.We found that p-ERK1/2 was appeared in the hucMSC-Exs and increased in NSCs when treating with hucMSC-Exs.ERK1/2 pathway inhibitor PD98059 can block the posistive role on the proliferation and differentiation of NSCs.Conclusion:On one hand,hucMSC-Exs promoted functional recovery of hind limb and repaired of the transverse spinal cord injury in rat models,limited the lesion size and inhibited glial cells activation,promoted neurogenesis and reconstruction of sypase,attenuated inflammation,enhanced the proliferation and differentiation of NSCs by targeting ERK1/2 pathway.On the other hand,hucMSC-Exs have no positive roles on migration of NSCs and inhibited the outgrowth of axonal fibers which were unbenifit to the repair of SCI.All the foundings illustrated that hucMSC-Exs enhanced the functional recovery of traumatic spinal cord injury rat by promoting neurogenesis and axonal regeneration rather than outgrowth of the axonal fiber。...