The Mechanism Of EGFR~+ Neural Stem Cell-derived Exosomes In Spinal Cord Injury Treatment

Objective:Spinal cord injury(SCI)is a severe traumatic disease of the central nervous system,characterized by damage to the structure and function of the spinal cord,resulting in loss of motor and sensory functions below the injury level,and even death.Currently,the clinical treatment methods for SCI are limited,and a safe and effective method is urgently needed to solve the worldwide problem.Stem cell exosomes therapy is an emerging safe and effective treatment method with important transformation prospects in the treatment of spinal cord injury at present.This study intends to explore the effect of EGFR~+neural stem cell exosomes on spinal cord injury reparation and its intrinsic mechanism.Methods:(1).EGFR~+neural stem cell subpopulations were sorted by flow cytometry and cultured in vitro;The functional characteristics of EGFR~+neural stem cells were determined by neurosphere-assay and cell differentiation experiment.(2)Exosomes of EGFR~+neural stem cells were extracted and characterized by TEM(transmission electron microscopy),nanoparticle tracking analysis and Western Blot.(3)EGFR~+neural stem cell exosomes were used to treat spinal cord injury mice.Behavioral score,cortical evoked potential,neurogenic bladder assessment,and spinal cord histological staining were used to evaluate the neurological recovery of spinal cord injury mice.(4)miRNA-seq was used to screen the gene expression of EGFR~+neural stem cell exosomes,and q RT-PCR was used to confirm the gene expression.(5)Inhibit the expression of miR-34a-5p in EGFR~+neural stem cell exosomes,intervene in spinal cord injury mice,and determine the effect of exosomal miR-34a-5p on the neurological recovery of spinal cord injury mice by behavioral score,cortical evoked potential,neurogenic bladder evaluation,and spinal cord histological staining evaluation.(6)Dual-Luciferase Reporter Assay System,q RT-PCR,and Western Blot were used to find the neuronal targets of miR-34a-5p.(7)Construct Hdac6 overexpression plasmid,and explore the mechanism of miR-34a-5p downstream target by immunofluorescence staining and Western Blot.(8)Small molecule inhibitors of HDAC6 were used to intervene in spinal cord injury model mice,and behavioral score,cortical evoked potential,neurogenic bladder assessment,and spinal cord histological staining were used to confirm that exosomes regulate downstream HDAC6 through miR-34a-5p to play a role in the neurological recovery of spinal cord injury mice.Results:(1)EGFR~+neural stem cells were successfully selected,and EGFR~+neural stem cells had stronger proliferative activity and neural differentiation characteristics;(2)Exosomes of EGFR~+neural stem cells were successfully extracted,and the exosomes were characterized by electron microscopy,particle size,and protein markers.(3)EGFR~+neural stem cell exosomes can promote spinal cord injury repair,and the effect is better than that of primary culture neural stem cell exosomes.(4)miRNA-seq was used to obtain miRNA component expression profiles of EGFR~+neural stem cell exosomes,in which miR-34a-5p had a high abundance in EGFR~+neural stem cell exosomes,and showed different expression in EGFR~+NSCs-Exos treated neurons.(5)After the expression of miR-34a-5p in EGFR~+NSCS exosomes was inhibited,the therapeutic effect of EGFR~+NSCS exosomes on spinal cord injury became worse.(6)EGFR~+neural stem cell exosomes can reduce the expression of HDAC6in neurons,and HDAC6 has binding sites with miR-34a-5p.(7)After HDAC6 overexpression in neurons,EGFR~+neural stem cell exosomes decreased their ability to promote axon growth,and EGFR~+neural stem cell exosomes increased microtubule acetylation level and decreased microtubule tyrosine level by inhibiting HDAC6 expression.(8)Based on inhibiting the expression of miR-34a-5p in EGFR~+neural stem cell exosomes,SW-100 a small molecule inhibitor of HDAC6 can restore the deterioration of exosomes efficacy caused by miR-34a-5p inhibition.Conclusion:Exosomal miR-34a-5p of EGFR~+neural stem cells can be derived to neurons,improve neural microtubule stability and promote the growth of neurites via Hdac6 downregulating,and thus promote the recovery of nerve function after spinal cord injury.