| BackgroundThe spinal cord is an important part of the central nervous system.Spinal Cord Injury(SCI)is a serious central nervous system injury.Patients with spinal cord injury will show varying degrees of motor and sensory disturbances.The mild ones can recover after several months of treatment,but the severe ones will develop into incomplete paraplegia or even paraplegia,which greatly affects the quality of life,and even has serious complications and crises.life.However,the high incidence of SCI and high treatment costs have greatly threatened the overall health condition of patients,and brought heavy treatment burdens and economic costs to the patients’ families.For more than half a century,various treatment methods such as drugs,surgery,hyperbaric oxygen and physical therapy have been used in the clinical treatment of SCI,but none of them have achieved satisfactory results.Until today,the repair of spinal cord nerves after SCI is still a major challenge and clinical problem.In recent years,the therapeutic effects of stem cell transplantation have been extensively studied and proved to have broad application prospects.In the process of stem cell transplantation,the paracrine function of stem cells plays a key role.Exosomes are extracellular vesicles with a diameter of approximately 10-100 nm.Many types of tissues and cells can release exosomes,so exosomes exist in a variety of tissue fluids,secretions,and cell culture fluids.Exosomes can mediate cell-cell communication by delivering biologically active molecules,including DNA,mRNA,miRNA,proteins,lipids,etc.The exosomes released by mesenchymal stem cells(MSC-Exos)can attenuate cell apoptosis,inflammation and promote angiogenesis after SCI.In this paper,by extracting exosomes derived from human umbilical cord mesenchymal stem cells,in vivo experiments and in vitro experiments were used to verify the treatment of spinal cord injury,verify its therapeutic function,and detect the expression and effect of p-AKT in it.MethodsWharton’s jelly of the human umbilical cord was used to extract hUCMSCs through enzymatic digestion method.The hUCMSC-Exos was extracted from the culture medium by ultracentrifugation.Transmission electron microscopy,Zetasizer Nano particle size analysis,and Western Blot were used to detect the morphological characteristics,particle size and molecular markers of hUCMSC-Exos.Allen’s spinal cord strike model was used and treated with hUCMSC-Exos.The BBB score scale was used to evaluate the recovery level of the hind limbs of rats,HE staining and immunohistochemical staining were used to analyze the degree of tissue degradation and protection of spinal cord injury,as well as the expression level and distribution of the expression of p-AKT(Ser473)in tissues of spinal cord injury.The BV-2 cell line and the compound hydrogen peroxide were used to construct a cell oxidative damage model,and hUCMSC-Exos was used for treatment.The reactive oxygen probe is used to detect the concentration of reactive oxygen by measuring the signal of fluorescence in the cell to determine the degree of damage.Western Blot was used to analyze the expression level of p-AKT.The p-AKT agonist SC79 and inhibitor MK-2206 were used to treat oxidative injury models to explore the relationship between p-AKT signaling pathway and exosomes in the treatment of spinal cord injury.ResultshUCMSC-Exos showed a typical cup holder-like structure under TEM.The diameter of exosomes analyzed by Zetasizer Nano is 73.4±8.6 nm.Western Blot analysis showed a typical marker of CD63(+),TSG101(+),Calnexin(-).In in vivo experiments,the hind limb motor function of rats was immediately lost after the model was constructed.As time passed,the hind limb motor function gradually recovered.The hUCMSC-Exos-treated rats recovered more significantly than the control group.HE staining showed that the spinal cord tissue of rats treated with hUCMSC-Exos was less damaged.Immunohistochemical staining showed that the spinal cord tissue of rats treated with hUCMSC-Exos showed more p-AKT expression than the control group.In the in vitro experiment section,the active oxygen fluorescence signal in the cell was stronger after the model was constructed,suggesting cell damage conducted by hydrogen peroxide.After hUCMSC-Exos treatment,the cellular reactive oxygen signal was lower than that of the control group.Western Blot indicated that the expression of p-AKT increased.SC79 can reduce the cellular reactive oxygen signal,and MK-2206 can prevent hUCMSC-Exos from reducing the reactive oxygen signal.Western Blot results indicated that p-AKT(Ser473)expression increased after SC79 treatment,and MK-2206 could prevent the increase of p-AKT(Ser473)expression and reverse the effect of hUCMSC-Exos in promoting the increase of p-AKT(Ser473)expression.ConclusionExosomes derived from human umbilical cord mesenchymal stem cells can promote the repair of the spinal cord in rats and the recovery of hind limb motor function,protect the injured spinal cord tissue,and inhibit the formation of cavities.It can reduce the reactive oxygen intensity of oxidative damage of BV-2 cell line.hUCMSC-Exos may promote the proliferation and repair of glial cells through the p-AKT pathway,and then form a new signal transduction pathway at the injury site to play a role in the repair of spinal cord injury. |
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