| Objective:To study the effect of intravenous injection of Neuroglobin(Ngb)transfected Adipose-derived stem cells(ADSCs)combined with repaglinide on rat injury,and to provide a theoretical basis for clinical treatment of spinal cord injury.Methods:1.Take a 3-month-old healthy SD rat,extract ADSCs from the adipose tissue in the inguinal area,and subculture the cells to the third generation,test the surface progeny of the passaged cells by flow cytometry,condense and freeze them after identified as ADSCs.Amplify Ngb by polymerase chain reaction(PCR)and make it successfully connected to the lentiviral vector pLenti6.3-IRES-EGFP,and then transfected into 293T cells,and finally the virus solution was transfected into ADSCs.Fluorescence microscope was used to test the expression of GFP,and WB was used to test the expression of Ngb in ADSCs.Use superparamagnetic iron oxide nanoparticles(SPIONs)to label ADSCs successfully transfected with Ngb to test the transplantation efficiency and survival rate of ADSCs.2.A modified Allen s method was used to establish 48 rat spinal cord injury models,which were randomly divided into blank control group,repaglinide group,transfected stem cell group,transfected stem cell+repaglinide(which is combined group).Both the transfected stem cell group and the combined group were intravenously injected with 0.3 ml of SPIONs labeled Ngb-transfected ADSCs(1×10~7/ml),once per day for 6 days.The combined group and repaglinide group were given gavage(0.4 mg/kg)once a day for 6 days.The blank control group was injected with the same amount of DMEM medium.3.The experimental rats in each group were scored by the injury behavior(BBB)method on the 1d,3d,5d,1w,2w and 4w,and HE staining and transmission electron microscopy observation were performed at 1w and4w respectively.And the expression of Ngb in spinal cord tissue was detected by Western blot.Results:1.Extraction and identification of ADSCs ADSCs were successfully isolated from the inguinal adipose tissue of SD rats.After subculture,the expression levels of cell surface antigen CD49d and CD44 were 47.21%and 50.05%respectively by flow cytometry.The expression of CD14 and CD45 was 0.72%and 2.57%,respectively.2.Ngb tracing Ngb-transfected ADSCs were found to have obvious GFP expression when they were cultured to day 3,animal experiments performed with Ngb-transfected ADSCs 5 days after culture,and stem cells were transfected at 1W,2W and 4W whose expression of GFP was detected in the spinal cord tissues of the rats in the group,suggesting that Ngb was expressed at the site of spinal cord injury,and the expression gradually increased with time.3.Survival results of SPIONs labeled ADSCs The survival rates of SPIONs labeled ADSCs group on the 1st,3rd,and 5th days were 96.12±0.75%,95.35±1.24%,95.61±1.35%,and the unlabeled ADSCs group on the 1st,3rd and 5th day the survival rates were 95.43±1.12%,96.45±1.70%,and 96.10±0.12%,respectively,No statistical difference between the two groups(P>0.05).4.BBB score After 3 days of modeling,the BBB(Basso-Beattie-Bresnahan)score of each group of rats was 0-2 points,and the four groups were compared in pairs.The difference was not statistically significant(P>0.05),indicating successful modeling;At 5d,7d,14d,and 28d after the model,the BBB score trend of the rats in the four groups was always:the blank control group was lower than the repaglinide group,the repaglinide group was lower than the transfected stem cell group,while the combined group had the highest,with the two groups compared with two another in four groups,the difference was statistically significant(P<0.05).5.HE staining results At 1w,different levels of inflammatory cell and fibroblast infiltration,tissue disorder,vacuole-like changes of nerve cells and increased interval of residual tissues appeared in the spinal cord tissue of each group of rats,among them the obvious one was the blank control group while the infiltration of inflammatory cells in the repaglinide group was relatively small as well as the number of neuronal vacuolization.The pathological changes in the transfected stem cell group and the combined group were the least.At the 4th week,the spinal cord tissue of each group of rats showed various degrees of tissue disorder,glial scar formation,and neuronal vacuolization.The blank control group was more obvious than the repaglinide group,and the repaglinide group was more obvious than the transfected stem cell group.The joint group changed the least.6.Electron microscopy results At the first week,changes in edema adhesion,increase of synaptic space and decrease of synapse in the spinal cord tissue of rats in each group were observed.At the 4w,the demyelinating lesions of the nerve tissue worsened,some of the myelin sheath disappeared,neuronal cell lysis and the number of glial cells increased.The observation results of the above two time points showed that the blank control group changed significantly than the repaglinide group.While in the repaglinide group,the change was more obvious than in the transfected stem cells,and the combination group had the least change.7.Results of WB test of Ngb The expression of Ngb in blank control group,repaglinide group,transfected stem cell group and combined group showed as 0.08±0.04,0.09±0.03,0.41±0.10,0.45±0.09 at 1w,and Ngb at 4w its expression levels were 0.12±0.04,0.15±0.02,0.61±0.08,0.72±0.12 respectively.Compared with the combination group,the empty control group and the repaglinide group at two time points had statistically significant differences(P<0.05).The combined group had the most Ngb expression and the best neurological recovery.Conclusion:Intravenous injection of Ngb-transfected ADSCs can target migration to the site of spinal cord injury,and has a certain effect on rat spinal cord injury,the treatment combined with repaglinide is more obvious. |
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