The Study On Promoting Axon Regeneration Of Cortical Neurons By Schwann Cell Exosomes

?Objective?Spinal cord injury(SCI)is a severe traumatic diseases,which could lead to high disability.But its repair is still very difficult.Our group have confirmed that transplantation of Schwann cells(SCs)can promote the restoration of function by improving the microenvironment in lesion site.However,it still has several limitations,such as ethics and immune rejection,which really impede its translation from basic research to clinic.Exosomes,as a better alternative to cell transplantation,has a promising future.Thus,this study is aimed to explore the mechanisms how the Schwann cell derived exosomes(SCDEs)promote axon regeneration by improving microenvironment,which may have a huge potential to be used in clinic.Moreover,it will give a novel direction and basic theory for the non-cell repair stradege in spinal cord injury.?Method?1.Isolation,culture and identification of Schwann cells: separate Schwann cells from the sciatic nerve of Wistar rats.The cells will be cultured and purified with basal medium,purified medium and proliferative medium.Culture the cells to the 3rd to 5th generation.Use S-100 fluorescence staining for identification.2.Isolation and identification of Schwann cells exosomes: cell culture supernatant(media with exosome-free serum)from the 3rd to 5th generation Schwann cells was applied to extract exosomes by Beckman ultracentrifugation.The morphology and distribution of exosomes were detected by scanning electron microscope(SEM);the diameter of exosomes was determined by nanoparticle tracking analysis(NTA)and Western Blot was used to detect specific markers.3.The effect of Schwann cells exosomes on axons: Coculture different concentrations of Schwann cell exosomes and 30 ng/ml proBDNF with cortical neurons,observing its biological functions,such as cell morphology,survival and the growth of axons.The axons of each group was measured by Image J to find out the optimum concentration of exosomes.Then under this situation,the neurons were detected by immunofluorescence and cell dynamic observation.The fluorescence images were detailed by HCA-Vision Software,such as the length of the total neurites,the length of the longest neurites,the number of neurite branches,and the number of neurite roots.4.RhoA/ROCK signalling detection: Western Blot was used to detect the level of total RhoA,GTP-RhoA,ROCK II and p-CRMP-2,which are related to RhoA/ROCK pathway.?Result?1.The feature of Schwann cells and its exosomes: After purification,there are only a few fibroblasts among Schwann cells,which showed the purity of Schwann cells is over 95%.With improved extraction protocol,SCDEs were cup bottom shaped under the SEM,NTA analysis showed that its diameter was between 40 and 180 nm,with an average of 106.5 nm,the specific protein markers CD9 and Alix were positive,and control protein TSG101 negative.What's more,p75 NTR was also in high expression.2.SCDEs promote axon regeneration: Compare with Con group,proBDNF could lead to the apotosis and axon retraction in 4h and 20 h.Detect the cell survival and axon length at 4h,the mean survival of cortical neurons is 77.4% and the mean axon length is 63.4 nm in proB group,which is much less than Con group.We also found that proBDNF could induce axon retraction in Biostation system.These results showed the apotosis and axon retraction model induced by proBDNF has been successfully made.In further experiments,10 ug/ml SCDEs can significantly increase the total neurite length(448.87 um),the max neurite length(98.53 um)and the number of neurite branches(19.4)(p<0.05).However,it had no effect on the number of neurite roots(p>0.05).3.RhoA/ROCK signalling detection: The expression levels of GTP-RhoA,ROCK II and p-CRMP-2 were significantly higher than those in the control group(p<0.05).However,compared with proB group,GTP-RhoA,ROCK II and p-CRMP-2 was lower in proB+Exo group(p<0.05).There was no significant difference in total RhoA expression among the four groups(p>0.05).?Conclusion?1.The platform of SCDEs has been successfully established.The extraction scheme of Schwann cells and SCDEs were optimized,which could stably and efficiently extract the exosomes from Schwann cells.It can serve as a base for the research of SCDEs in the central and peripheral nervous system.2.This study showed that SCDEs could reverse growth cone collapse induced by proBDNF to promote axon regeneration.It will be a novel theoretical basis for exosomes to promote axonal regeneration by improving the microenvironment,especially for glia cells.3.The relationship between the Schwann cells and RhoA/ROCK signaling pathway was clarified.To further study the mechanisms how SCDEs promote axon regeneration could promote the development of repair of central nervous system from cell transplantation to non-cell strategy,which will offer a novel direction for clinical repair of spinal cord injury.