Neuroprotective Effect Of NGF Gene Modified Neural Stem Cells On Spinal Cord Injury

Background:Spinal cord injury(SCI)usually leads to severe motor,sensory and autonomic nervous system dysfunction,and its high disability rate seriously affects the normal life of patients.There is still no effective treatment.Neural stem cells(NSC)have multidifferentiation potential,which can supplement the loss of neuronal cells and secrete neurotrophic factors to protect the neural microenvironment.Therefore,this study used neural stem cells to overexpress nerve growth factor(NGF)gene to explore its repair effect on spinal cord injury.Objective:In this study,NGF gene-modified neural stem cells and 3D hydrogels were combined to treat spinal cord injury in mice.To explore the effect of combined therapy on nerve tissue regeneration and functional recovery after spinal cord injury,and to provide new ideas for the treatment of spinal cord injury in the future.Methods:1.The primary NSC of mice was extracted,the proliferation ability was detected by CCK8,and the expression of NSC marker Nestin was detected by immunofluorescence.The expression of NGF was detected by ELISA,q RT-PCR and Western blotting.CCK8 and immunofluorescence were used to detect NSC activity and stemness.After 7 days of differentiation culture,NSC differentiation was detected by immunofluorescence.2.NSC was cultured in 3D hydrogel and divided into NSC group,NSC+gel group and NSC-NGF+gel group.CCK8 and immunofluorescence were used to detect the effect of NGF on the proliferation and differentiation of NSC in hydrogel environment.3.The biocompatibility of hydrogel was detected by hemolysis test and HE staining.Eight-week-old C57BL/6J female mice were selected to establish spinal cord injury models and divided into Sham group,SCI group,SCI+gel group,SCI+NSC-GFP+gel group and SCI+NSC-NGF+gel group.Cells were transplanted in situ,BMS score was used to evaluate the recovery of motor function,and HE staining was used to observe the morphology of spinal cord tissue.Apoptosis was detected by TUNEL staining and Western blotting.The expression of NGF was detected by ELISA and q RT-RCR.The expression of neural differentiation-related proteins was detected by immunofluorescence and Western blotting.Results:1.The extracted primary NSC showed spherical growth and high expression of Nestin.Overexpression of NGF lentivirus infected NSC,found that NGF expression increased,while the activity and stemness of NSC were not affected.After 7 days of differentiation culture,immunofluorescence results showed that LV-NGF induced NSC to differentiate into neurons and inhibited its differentiation into astrocytes.2.Calcein / PI staining showed that the hydrogel had good cell compatibility.CCK8 assay showed that the hydrogel environment was more conducive to the proliferation of NSC-NGF.After 7 days of differentiation culture,immunofluorescence results showed that the hydrogel environment was more conducive to the differentiation of NSC-NGF into neurons.3.The results of hemolysis test and HE staining showed that the 3D hydrogel had good biocompatibility.After 28 days of spinal cord injury model treatment,q RT-PCR,immunofluorescence staining and Western blotting results showed that hydrogel-loaded NSC-NGF could continuously express NGF at the injured site,inhibit cell apoptosis and astrocyte proliferation at the injured site,promote neuronal regeneration,endogenous myelin regeneration and endogenous neurogenesis around the injured segment,improve hindlimb motor function in mice,and promote tissue repair.Conclusions:Combined treatment of NGF gene-modified neural stem cells and hydrogel in mice with spinal cord injury has a neuroprotective effect by inhibiting apoptosis and astrocyte proliferation and promoting neuronal survival and regeneration.