| Objective:Central nervous system(CNS)diseases are a group of serious diseases,including diseases of the brain and spinal cord.These diseases can cause damage to the function of the nervous system,thus affecting People’s Daily life.Spinal cord injury(SCI)is a kind of very harmful central lesion,it may be caused by trauma,will cause serious damage to the patient’s life,including the weakened physical movement ability,the loss of sensory function,etc.Because of the difficulty in treating SCI,there is an urgent need for new treatments.In recent years,neural stem cells have been widely studied as a new therapeutic method.Neural stem cells have powerful regenerative and differentiation capabilities,and can help rebuild the neural network of spinal cord injury and restore motor and sensory functions by differentiating into daughter cells including neurons,astrocytes and oligodendrocytes,providing effective treatment for diseases.Although neural stem cell therapy SCI has a broad prospect,there are still many problems in its theoretical and practical application,such as the mechanism of neural stem cell transformation into neurons,how to improve its transformation efficiency,and the lack of clinical data on neural stem cell therapy.In recent years,it has been found that astrocytes(AS)and their extracellular vesicles(EVs)have many advantages,such as easy access and storage,high biocompatibility and safety,good tissue permeability and intracellular transport ability,not limited by the blood-brain barrier,and excellent transport vector function.This study aims to explore whether the extracellular vesicles(AS-EVs)secreted by astrocytes can promote the growth and differentiation of neural stem cells,so AS to make up for the loss of neurons after SCI,providing new ideas and possibilities for the treatment of spinal cord injury.Methods:(1)The brain astrocytes(AS)of SD rats were cultured by pancreatic enzyme digestion method,and the morphologic characteristics of AS were observed by microscope and immunofluorescence staining techniques of specific marker genes were used to identify the obtained AS.In addition,the particle size of AS-EVs can be observed by transmission electron microscopy,and the expression level of specific marker proteins can be detected by western blotting to identify the extracted AS-EVs more accurately.(2)The brain neural stem cells of SD rats were cultured by pancreatic enzyme digestion method,and the cell morphology and specific markers were identified by immunofluorescence staining under microscope.(3)The cultured and purified third-generation neural stem cells were subjected to group intervention: Group 1 was added with 1 mg/m L concentration of AS-EVs for intervention culture for 6 days,group 2 was added with the same volume of PBS for intervention culture for 6 days.(4)Immunofluorescence,qRT-PCR and Western blot were used to detect the expression levels of neuronal markers after PBS and AS-EVs intervention,respectively.Results:(1)Microscopically,the astrocytes appear as irregular stars,and immunofluorescence responses show high levels of the signature protein GFAP expression.The morphology of AS-EVs was round or oval cup,and the aperture of AS-EVs was about 10-200 nm AS measured by dynamic particle radiometry.Western blotting also proved that CD63 and TSG101 markers were positive.(2)Microscopically,the neural stem cells were spherical and distributed in clumps.The immunofluorescence reaction showed high expression of CD133 and Nestin.(3)Compared with PBS group,the differentiation of neural stem cells in AS-EVs group was more obvious,the cell morphology was more plump,there were obvious prominents on the surface,and the neural stem cells were mainly neurons,and the expression levels of tubulin β3 antibody and neurofilament protein 200 antibody were also higher(P <0.05).Conclusions:Experiments have shown that AS-EVs can effectively promote the differentiation of neural stem cells into neurons,thus helping to replace the loss of neurons in central diseases(such AS spinal cord injury),and thus promote the regeneration and repair of the nervous system. |
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