| BackgroundSpinal cord injury is a kind of severe traumatic disease of central nervous system,which causes incomplete or complete loss of voluntary motor and sensory function,and is a serious health hazard.At present,there is no effective treatment for spinal cord injury in clinical practice.In recent years,traditional spinal cord injury treatments represented by surgery,medication and rehabilitation and novel spinal cord injury treatments represented by stem cell transplantation and tissue engineering repair have made some progress,but still cannot achieve satisfactory clinical effects of nerve repair.Therefore,the research on spinal cord injury repair still needs to be further carried out,and it is very important to find safe,efficient and more promising nerve repair drugs to promote the research on spinal cord injury repair and regeneration.Platelet-rich plasma(PRP)is a kind of biological agents derived from autologous or allogeneic blood,which is rich in a large number of bioactive factors and has excellent tissue repair and regenerative potential.Studies have shown that PRP not only releases a large number of bioactive factors to participate in the process of tissue repair,but also secretes a large number of extracellular vesicles(EVs)that also play an important role in the tissue repair process and even have a superior tissue repair potential than PRP.Current studies have confirmed that PRP has a beneficial effect on repair and regeneration after spinal cord injury,but there are still some limitations.PRP-derived EVS(PRP-EVS)has higher concentration of growth factors,better stability and biocompatibility,and lower immunogenicity,so it is expected to be a more promising material for spinal cord injury repair than PRP.Therefore,this study was conducted to investigate the role of PRP-EVs in repair and regeneration after spinal cord injury and to reveal their related mechanisms.ObjectsIn this study,rat PRP-EVs and human PRP-EVs were extracted from rat blood and human peripheral venous blood,respectively,and both were characterized and identified,aiming to confirm that both rat PRP and human PRP contain large amounts of EVs.In addition,the effects of PRP-EVs on the proliferation,migration and differentiation umbilical vein endothelial cells(HUVECs)and neural stem cells(NSCs)were further observed in vitro,the effects of PRP-EVs on tissue repair and the recovery of nerve functions after spinal cord injury were also observed in vivo,and the related mechanisms were revealed.This study will look for a promising new potential substance for the repair and regeneration of spinal cord injury.Methods1.The PRP were extracted from rat blood and human peripheral venous blood using "two-step centrifugation method" and activated by calcium chloride solution.Finally,the rat PRP-EVs and human PRP-EVs were extracted using EVs extraction kit.They microscopic morphology was observed by transmission electron microscopy,the particle size was identified by NTA,and the surface marker proteins were identified by Western blot.Besides,ELISA method was also applied to detect the concentrations of important growth factors contained in rat PRP and PRP-EVs to reveal a possible mechanism for the superiority of PRP-EVs over PRP in the field of tissue repair.2.The fluorescent labeling method was used to observe the uptake of PRP-EVs by HUVECs cells,the CCK-8 method was used to detect the effect of PRP-EVs on the proliferation of HUVECs cells,the live-dead cell staining was used to detect the effect of PRP-EVs on the apoptosis of HUVECs cells,the scratch assay and Transwell assay were used to detect the effect of PRP-EVs on the migration of HUVECs cells,and the tubule formation assay was applied to detect the effect of PRP-EVs on the angiogenesis of HUVECs cells in vitro.3.The fluorescent labeling method was used to observe the uptake of PRP-EVs by NSCs cells,CCK-8 method and Ki67 immunofluorescence staining were used to detect the effect of PRP-EVs on the proliferation of NSCs cells,live-dead cell staining was used to detect the effect of PRP-EVs on the apoptosis of NSCs cells,Transwell assay was applied to detect the effect of PRP-EVs on the directional migration of NSCs cells,and cellular immunofluorescence method was used to detect the effect of PRP-EVs on the neural differentiation of NSCs cells.4.Constructed a rat clamped spinal cord injury model,BBB score,hind limb motor footprint analysis and hind limb neurophysiological assay were used to evaluate the effect of PRP-EVs on neurological recovery after spinal cord injury,HE staining,Masson staining and tissue immunofluorescence staining were used to evaluate the effect of PRP-EVs on tissue repair and regeneration after spinal cord injury.Results1.Both rat PRP and human PRP contained a large number of PRP-EVs,and the results of transmission electron microscopy showed that both rat PRP-EVs and human PRP-EVs were "disc-shaped" or "cup-shaped" with a diameter of nearly100 nm.The NTA results showed that the particle size of rat PRP-EVs was 121.6±46.8 nm and that of human PRP-EVs was 119.2±52.1 nm,which were within the size range of EVs.The Western blot results showed that rat PRP-EVs with high expression on EVs marker proteins Alix,TSG101 and CD9,human PRP-EVs with high expression on EVs marker proteins Alix,TSG101,CD9 and CD63.Moreover,the result of ELISA showed that PRP-EVs with the same total protein concentration contained a higher concentration of many growth factors than PRP.2.HUVECs cells can phagocytose PRP-EVs into the cells which affect their biological functions.In addition,PRP-EVs can effectively inhibit hypoxia-induced apoptosis of HUVECs cells and promote the proliferation,migration and angiogenic differentiation of HUVECs cells.3.NSCs cells can also phagocytose PRP-EVs into the cells which affect their biological functions.In addition,PRP-EVs can effectively inhibit hypoxia-induced apoptosis in NSCs cells,improve the tolerance to hypoxic environment,promote the proliferation and directed migration of NSCs cells.PRP-EVs can also promote the differentiation of NSCs cells,but this kind of differentiation is not directed.4.PRP-EVs can effectively reduce the secondary spinal cord injury,induce local angiogenesis,promote the repair and regeneration of neural tissue and the recovery of related neural functions after spinal cord injury.Conclusions1.The "two-step centrifugation method" method is an effective method for the preparation of PRP,which can successfully isolate PRP from rat blood and human peripheral blood.In addition,EVs extraction kit can effectively extract standard EVs from rat PRP and human PRP.Besides,in terms of many important growth factors,rat PRP-EVs are enriched with a higher concentration than PRP.2.PRP-EVs can inhibit the apoptosis and promote the proliferation,migration and angiogenic differentiation of HUVECs cells.3.PRP-EVs can inhibit the apoptosis and promote the proliferation,directed migration and neural differentiation of NSCs cells.4.PRP-EVs can promote tissue repair and regeneration and promote the recovery of related neurological functions after spinal cord injury.InnovationIn this study,we investigated the effects of RPR-EVs,a novel PRP derivative,on tissue repair and neurological recovery after spinal cord injury.The results showed that RPR-EVs could promote tissue repair and related neurological recovery after spinal cord injury through the dual pathway of inducing local vascularization and neural differentiation,and tentatively demonstrated that RPR-EVs might be a more promising potential material for spinal cord injury repair.No relevant studies have been published after scientific novelty-checking which means that this study has original innovation. |
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