Effects Of Neural Stem Cell And Nerve Neurotrophic Factor 3 Gene Modified Olfactory Ensheathing Cell Co-transplants On Functional Recovery After Traumatic Brain Injury In The Adult Rat

BackgroundTraumatic brain injury(TBI)is a serious traumatic disease of the central nervous system with high incidence rate,high disability rate and high mortality.However,patients often leave refractory neurological dysfunction in the later stage of injury.In recent years,cell transplantation provides a therapeutic strategy for the repair of nerve injury.Neural stem cells(NSCs)are cells with self-renewal and differentiation,which can survive,differentiate and replace damaged or apoptosis neurons after transplantation.Olfactory ensheathing cells(OECs)are cells that exist in the olfactory system and have the ability of life-long regeneration and division.And they can secrete a variety of neurotrophic factors,which contribute to the growth of damaged axons and the regeneration of myelin sheath.Neurotrophin-3(NT-3)can promote the differentiation and extension of axons,has the function of anti-apoptosis,mediates cell survival,and induces stem cells to differentiate into neurons in the process of repair after nerve injury.ObjectiveIn this study,NT-3 gene modified olfactory ensheathing cells(OECs-NT-3)were constructed.Then NSCs and OECs-NT-3 co-transplantation were performed on rats after traumatic brain injury.To observe the effects of NSCs and OECs-NT-3 co-transplants on functional recovery after traumatic brain injury in the ratMethods1.Primary culture OECs and NSCs,and cell identification.2.Construction of NT-3 gene modified olfactory ensheathing cell:An appropriate amount of OECs were seeded into a 6-well plate.The lentiviral vector stock solution containing NT-3 gene(1×10~9 TU/ml)were diluted with a MOI value of 100,10,1,0 by culture medium.The virus gradient diluent were put in 6-well plate,and cultured in 37?incubator for 24 hours,then changed the fresh medium.48 hours later,the staining effect was observed under fluorescence microscope.And cell culture supernatants were collected at 2 d,7 d,14 d,and 28 d after transfection in each group,and the NT-3 content in the supernatants of each group was detected by ELISA.3.Experimental group:50 2-month-old male SD rats weighing(200±20)g were randomly divided into 5 groups:sham operation group(group A),TBI model group(group B),NSCs transplantation group(Group C),OECs-NT-3 transplantation group(Group D),OECs-NT-3 combined with NSCs transplantation group(Group E),10 rats in each group.4.TBI model of rat and cell transplantation:The modified Feeney's method was used to make TBI model.Cell transplantation was given on the 3rd day of modeling.NSCs cells were co-cultured with 5-bromodeoxyuracil BrdU(10?mol/L)for 72 h before transplantation.5.Modified neurological severity score in rats:Modified neurological severity scores(NSS)were performed on rats of each group at 1,7,14 and 28 days after cell transplantation to determine the degree of brain function impairment in rats.The lower the score,the better the nerve function.6.Hematoxylin-eosin staining and cell density calculation:Hematoxylin and eosin staining were performed.After sealed the glass slides,cell morphology and tissue structure were observed under microscope,and cell density was calculated.7.Immunohistochemistry:Immunohistochemical staining with P75 and BrdU antibody was performed,then the survival and distribution of NSCs and OECs were observed.8.TUNEL cell apoptosis detection:Apoptosis was detected in the damaged of focal.The apoptosis index was calculated.9.Statistical methods:SPSS 21.0 statistical software was used for analysis.Measurement data were expressed as Mean±standard deviation(Mean±SD).Comparisons among multiple groups were analyzed by one-way analysis of variance.Pairwise comparisons were performed by LSD-t test.The difference was statistically significant with P<0.05.Results1.Immunofluorescent staining of P75 antibody was performed to confirm that the cells were OECs.Immunofluorescent staining of Nestin antibody and neural stem cell induction differentiation tests were performed to confirm that the primary cultured cells from hippocampus were NSCs.2.The transfection effect was observed under fluorescence microscope after 48h.The results of ELISA showed that the content of NT-3 in the culture medium of OECs with MOI of 10 was higher than that of other groups,and it could be secreted continuously to 28days after transfection.3.The NSS score of rats:The motor function of the rats in the combined cell therapy group recovered significantly.The specific manifestations are:balance beam test stay time is more than 40 s or stable posture without falling,limb flexion recovery in tail lift test,ground walking gait tends to be stable or can recover straight walking,and abnormal movement disappears.NSS Scoring results:At 28 days,the combined group was different from the OECs-NT-3 group and the NSCs group.And the difference was statistically significant(P<0.05).4.Hematoxylin-eosin staining:At 28 days after transplantation,In the model group,the tissue structure of the lesions was disordered,and obvious tissue fractures and voids appeared.The organization structure of the cerebral cortex area in the combination group was relatively regular,and there were many nerve cells with intact capsules and clear nucleoli.Compared with the model group,OECs-NT-3 group,and NSCs group,the density of injured cells in the combined group was significantly increased,and the difference was statistically significant(P<0.05).5.P75 and BrdU immunohistochemistry:After transplantation for 28d,the positive staining results of P75 and BrdU were found in the cell treatment group,suggesting that the two kinds of transplanted cells can survive continuously and widely distributed around the lesion.6.Apoptosis rate in brain tissue of rats:At 7 days after transplantation,The apoptosis rate in combined transplantation group was significantly lower than that in model group and OECs-NT-3 group and NSCs group,the difference was statistically significant(P<0.05).Conclusion1.OECs can stably secrete NT-3 after being transfected with a lentiviral vector containing the NT-3 gene.2.Neural stem cells and NT-3 gene-modified olfactory ensheathing cells can survive for a long time after transplanting to the lesions of TBI model rats and gather at the edge of the injury,and promote the recovery of nerve motor function in rats.3.Transplantation of neural stem cell combined with NT-3 gene modified olfactory ensheathing cell can alleviate neuronal apoptosis after brain injury in rats,and promote the recovery of nerve motor function.