| BackgroundAfter traumatic brain injury（TBI）,changes such as neuronal necrosis,inflammatory response,and lack of neurotrophic factors may occur in relevant parts,which can lead to refractory neurological dysfunction in patients.The application of cell transplantation offers new hope for improving neurological dysfunction after brain injury.Studies have shown that neural stem cells（NSCs）combined with neurotrophic factor 3（NT-3）gene-modified olfactory ensheathing cells（OECs）transplantation can improve neurological impairment after TBI.ObjectiveTo explore the effect of NSCs combined with NT-3 gene modified OECs（NT-3-OECs）transplantation on the expression of NGF after TBI in rats.Methods1.Animal grouping:100 adult SD rats were randomly divided into five groups: sham group,TBI group,NSCs treatment group,nt-3-oecs treatment group and NSCs combined with nt-3-oecs treatment group.2.Primary culture of OECs and NSCs: NSCs and OECs were extracted and cultured from hippocampus and olfactory bulb of neonatal rat brain.3.Construction of OECs modified with NT-3 gene: lentivirus carrying NT-3 gene was transfected into OECs,and the content of NT-3 in the supernatant was detected by ELISA.4.Modeling and cell transplantation: the brain injury model was made by modified Feeney DM in each group.One day after modeling,cells were transplanted,and 10 ul of corresponding cell suspension or normal saline were injected into the injury focus.5.Rat neurological deficit score: according to the standard of rat modified neurological deficit score（m NSS）,scores were performed at 1,7,14,and 28 days after transplantation to evaluate the degree of neurological deficit in rats.6.HE staining: Rats in each group were anesthetized,perfused,decapitated,and the brain tissue was embedded in paraffin,and sliced for later use.HE staining was carried out according to the instructions,and the samples were observed and photographed under a microscope.7.ELISA assay:The rat brain tissue was taken 7 days after transplantation,and the expression of inflammatory factors IL-6,IL-1 βand NGF protein was determined by ELISA method.8.Immunohistochemistry: The expression of p75,Nestin and NGF protein in rat brain tissue 7 days after transplantation was detected by immunohistochemical staining.9.The statistical method was spss21 0 statistical software was used to analyze 6 experimental data.The counting data were expressed by mean ±standard deviation.The two groups were compared by t-test,and the multi groups were compared by one-way variance.When p < 0.05,the difference was considered to be statistically significant.Results1.The content of NT-3 in the medium of OECs transfected with lentivirus was higher than that of untransfected OECs.2.On the 28 th day after transplantation,the m NSS score of the combined group was lower than that of the TBI group,the NSCs group and the OECs group（P < 0.05）.3.On the 28 th day after transplantation,compared with TBI group,NSCs group and OECs group,the combination group showed relatively regular arrangement,reduced cell swelling,repaired cavities,and clear nucleoli.4.On the 7th day after transplantation,compared with model group,NSCs group and OECs group,IL-6 and IL-1 in the combined group βThe expression level decreased（P < 0.05）,and the content of NGF protein increased（P < 0.05）.5.After 28 days of transplantation,p75（labeled OECs）and nestin（labeled NSCs）positive cells were more widely distributed in the combined group.The positive results of NGF in the combined group were significantly higher than those in TBI group,NSCs group and OECs group（P < 0.05）.ConclusionThe combined transplantation of NSCs and NT-3-OECs can improve the expression of NGF protein,reduce the inflammatory response in the injured area,and promote the recovery of nerve function,thereby achieving neuroprotective and repairing effects. |
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