Exosomes Secreted By Endothelial Cells Enhance Neural Progenitor Cell Activity And Promote Neural Restoration In Acute MCAO

Objective: Related studies have confirmed that exosomes are involved in intercellular communication and regulate the relevant behavior of target cells.The data indicate that brain microvascular endothelial cells secrete exosomes.Therefore,this study aims to further investigate the effect of endothelial cell exosomes on the survival of neural progenitor cells,and further validate it in the MCAO model,thus providing new ideas and potential clinical treatments for neurological function reconstruction in post-stroke injury areas.Methods: 1.Wild-type mouse neural progenitor cells(NPC)were isolated and cultured,and cultured with a special medium for stem cells,and the surface-specific antigen was identified by immunofluorescence after purification.2.The exosomes of mouse microvascular endothelial cells(bEnd.3)were extracted by ultracentrifugation and identified by transmission electron microscopy,Western blot and dynamic light scattering.NPCs were cultured at different concentrations,using CCK8 reagent,Edu reagent.The box was tested for its effect on the proliferation of NPCs;Trans-well,cell scratch assay was used to detect the effect of exosomes on the migration ability of NPCs;in the environment of nutrient factor deprivation,exosomes were treated for 3 h and detected by FACS/PI.The effect of apoptosis on NPCs was further examined by Western blot.3.After MCAO was successfully modeled,100 uL,100ug/mL exosomes or PBS were injected into the lateral ventricle by intracranial stereotaxic apparatus,and behavioral evaluation was performed on the 1st,3rd,7th,14 th and 21 st day respectively.The lesions of the animals were examined by brain magnetic resonance on day 1 and 21,and the volume of cerebral infarction was counted separately.Results: The neural progenitor-specific antibody Sox2 and Nestin double-labeled successfully identified the extracted cells;Edu and Dapi staining demonstrated that endothelial cells can promote the proliferation and migration of neural progenitor cells,and are proportional to the number of endothelial cells.Under the transmission electron microscope,the exosomes are elliptical or circular in shape and have a diameter of 30-150 nm.Western Blot assay showed that the expression of endothelial cell culture supernatant and exosomes TSG101,Alix and Flotillin-2 were positive.The extracellular vesicle size distribution obtained by dynamic light scattering analysis was in the range of 30-200 nm.In the conditioned medium to which the concentration of exosomes was 60 ug/mL,the proliferation and migration of NPCs were significantly promoted,and apoptosis was inhibited.The MCAO model was successfully established by TTC staining.The results of magnetic resonance imaging showed that the animals in the intracranial stereotactic injection group on the 1st and 21 st day after modeling were less infarct volume than the animals in the PBS group.The catwalk evaluation showed Animals in the exosomes group had better animal behavior than the PBS group.Conclusion: The exosomes of endothelial cells have the function of promoting the proliferation and migration of neural progenitor cells and inhibiting their apoptosis,and verifying the exosomes of endothelial cells in the middle cerebral artery occlusion model can improve the neurological function repair of animals after cerebral infarction.This may be a potential clinical treatment.