| ObjectiveThis thesis investigates the effects of extracellular vesicles?EVs?secreted by human umbilical cord mesenchymal stem cells?hUCMSCs?on the proliferation and migration of Schwann cells?SCs?,which provides novel ideas to explore a new direction in treatment of repairing peripheral nerve injury.In this study,extracellular vesicles?hUCMSC-EVs?from human umbilical cord mesenchymal stem cells were extracted and cultured with SCs.We find that hUCMSC-EVs are able to promote the proliferation and migration of SCs and promote repair of damaged nerves.Our work presents solid theory and practice to demonstrate an application of extracellular vesicles in tissue engineering that can help repair damaged peripheral nerves.Methods?1?Isolation and identification of hUCMSCs.?2?EVs?hUCMSC-EVs?were isolated and purified from hUCMSCs by ultra-high speed centrifugation.The morphological characteristics of EVs were observed by transmission electron microscopy.Expression of EVs marker proteins CD63 and CD9 was detected by Western blot.?3?Extraction and identification of Schwann cell?SCs?.?4?The effects of hUCMSC-EVs on proliferation and migration of SCs at different concentrations were detected by cck-8 and TranswellTM chamber experiments.?5?The endocytosis of EVs by primary SCs was observed via confocal microscopy.?6?hUCMSC-EVs were traced to the injured site of sciatic nerve,which showed that hUCMSC-EVs had a directional migration to SCs on rats.?7?The rat model of sciatic nerve transected injury was established and EVs were injected in vivo to observe the effect of EVs on SCs proliferation and migration in vivo.Results?1?The results of isolating hUCMSCs showed that hUCMSCs were spindle-shaped or edge-shaped.The surface markers of hUCMSCs were detected by flow cytometry,and the results showed that hUCMSCs were CD14,CD19,CD34,CD45 negative,CD73 and CD90positive.Furthermore,hUCMSCs could be induced to be positive for osteogenesis test.Lipid formation experiments produce lipid droplets.?2?The results of transmission electron microscopy showed that the hUCMSC-EVs had a circular or elliptic membranous structure.The particle size range was 80-650nm,with an average of around 168nm.Both the specific molecular markers CD9 and CD63 were expressed.?3?The primary SCs of newborn SD rats were cultured with uniform cell morphology,decent growth and spindle shape.After S100 and GFAP double fluorescence staining,the extracted cells were confirmed to be schwann cells.?4?Compared with the control group,hUCMSC-EVs significantly promoted the proliferation of primary SCs?P<0.05?and migration?P<0.05?,and showed a dose-effect relationship.?5?Cell membrane fusion occurred after hUCMSC-EVs were incubated with primary SCs.After co-incubation of DIO green fluorescent labeled hUCMSC-EVs and DAPI labeled rat SCs nuclei,confocal microscopy observation showed that the primary rat SCs could endocytosis hUCMSC-EVs.?6?DiR labeled hUCMSC-EVs were tracked on rats.After sciatic nerve injury,hUCMSC-EVs accumulated at the injury site and regulated its inflammatory response.?7?hUCMSC-EVs promoted the proliferation and migration of SCs in vivoConclusionshUCMSC-EVs can be applied to regeneration of peripheral nerve injury by facilitating the proliferation and migration of SCs. |
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