| Objective:Spinal cord injury(SCI)is the spinal cord structure and function injury caused by a various of causes,which results in obstacle of spinal cord nerve function,sensation,sphincter and plant nerve function.SCI can cause damage to neurons and glial cells,the interruption of neural pathway,neurological dysfunction,rapid proliferation of ependymal cells and abnormal differentiation of endogenous neural stem cells.Repeat transcranial magnetic stimulation(rTMS)is a technology using the role of time-varying magnetic fields on cerebral cortex to produce induced current to change the action potential of the cortical nerve cells.It can influence brain metabolism and nerve electrical activity.rTMS can excite nerve cells painlessly,depending on the intensity,duration and direction of stimulation.It can not only affect cortical nerve activity,but also has the continuous regulation effect on cortex.The frequency of rTMS can be increased or decreased to stimulate cortical nerve activity.Under proper parameters,rTMS can affect local blood flow in the brain,change local oxidative stress levels,and regulate nerve remodeling.In addition,research suggests that it has the ability to promote nerve regeneration.Stem cell therapy is a hot topic in medical research and has broad prospects in treatment for damage repair,especially in repair of nervous system damage.Currently,cells that are used for transplantation are neural stem cells derived from embryonic stem cell differentiation,neural stem cells extracted from fetal neural tissue,olfactory sheath cells,schwann cells and so on.Mesenchymal stem cells have wide potential for differentiation.In vitro culture,they can differentiate in different directions under different induction conditions.They can not only differentiate into osteoblasts,fat cells,chondrocytes and muscle cells,but also differentiate across the layer.Mesenchymal stem cell(MSCs)obtained from cord blood have a comparative advantage.Human umbilical blood MSCs are more primitive and have more differentiation and proliferative ability.Moreover,cord blood is collected from the placenta and umbilical cord stump after delivery,and the process is simpler than that from bone marrow or embryonic stem cells.Protected by placenta,its components are less likely to be contaminated by viruses and bacteria and the surface of umbilical cord blood stem cells has not yet formed blood group antigen and human leukocyte antigen,which does not cause obvious host resistance to graft.This experiment is to improve the motor function of rats after SCI by stimulating motor area of cerebral cortex and the combination of human umbilical cord blood mesenchymal stem cells.Methods:Adult male rats weighing 210-2 60g were selected and modified Allen's method was used for rat T10 spinal cord injury model.Rats were randomly divided into 6 groups:injury,injury+0.5 Hz rTMS,injury+10 Hz rTMS,injury+stem cells,injury+0.5 Hz rTMS+stem cells and injury+ 10 Hz rTMS+stem cells group.Day after surgery,injury+stem cells,injury+0.5 Hz rTMS+stem cells and injury+10 Hz rTMS+stem cells group were performed cord stem cell transplantation of spinal cord injury site.rTMS was performed in injury+ 0.5 Hz rTMS,injury+10 Hz rTMS,injury+ stem cells,injury+0.5 Hz rTMS+stem cells and injury+0 Hz rTMS+stem cells group.After surgery for 1,2 and 4 weeks,BBB behavioral evaluation and motor evoked potential(MEP)were performed for each group.HE staining was used to observe morphological changes of the spinal cord.Western blot was used to determine the expression changes of basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF).Immunohistochemical method was applied to determine the change of BrdU,Nestin,Tuj 1,Ng2 and GFAP.Results:.1.BBB score:BBB score in injury+0.5 Hz rTMS,injury+10 Hz rTMS,injury+stem cells,injury+0.5 Hz rTMS+ stem cells and injury+10 Hz rTMS+stem cells group was all higher than the injury group.The variation of each group was related to the time change,and the BBB score increased with time.There were significant differences in comparison between groups at different time points,and there were significant differences between different time points in each group.There was a significant difference between the injury group and the rest of the group at any time.The injury+0.5Hz rTMS+stem cell group and the injury+10Hz rTMS+stem cell group were significantly different from the rest of the group at any time.Moreover,BBB score of the injury+10Hz rTMS+stem cell group was significantly higher than that of the injury+0.5 Hz rTMS+stem cell group.2.MEP:The MEP the injury group was the longest,with the smallest amplitude and almost invisible.The incubation period in injury+0.5 Hz rTMS,injury+10 Hz rTMS,injury+stem cells,injury+0.5 Hz rTMS+stem cells and injury+10 Hz rTMS+stem cells group was short and the amplitude was long.Amplitude in injury+10 Hz rTMS group was longer than injury+0.5 Hz rTMS group.Aplitude in injury+10 Hz rTMS group and injury+stem cells group had no significant difference.Amplitude in injury+0.5 Hz rTMS+stem cell group and injury+10Hz rTMS+stem cell group were all higher than the other 4 groups.Aplitude in injury+10Hz rTMS+stem cell group was much higher than injury+0.5 Hz rTMS+stem cell group.3.HE staining:The lesion in injury group can be seen in the surface of the spinal cord.The spinal cord is obviously narrow,the structure of the spinal cord is loose,the cavity is formed and the neurons are dead.In injury+0.5 Hz rTMS,injury+10 Hz rTMS,injury+stem cells,injury+0.5 Hz rTMS+stem cells and injury+10 Hz rTMS+stem cells group,we can see spinal cord is loose,glial cells proliferate,and some nerve cells swell.Moreover,sinal cord tissue in injury+0.5 Hz rTMS+stem cells and injury+10 Hz rTMS+stem cells group was more loosened than injury+0.5 Hz rTMS,injury+10 Hz rTMS and injury+stem cells group.4.Western blot:EGF expression in injury+0.5 Hz rTMS,injury+10 Hz rTMS,injury+ stem cells,injury+0.5 Hz rTMS+stem cells and injury+10 Hz rTMS+stem cells group was obviously increased compared with injury group.There was no significant difference in EGF expression between the three groups.bFGF expression in injury,injury+0.5 Hz rTMS,injury+ 10 Hz rTMS,injury+stem cells had no obvious difference.bFGF expression in injury+0.5 Hz rTMS+ stem cells and injury+10 Hz rTMS +stem cells group was evidently increased compared with injury group.There was no significant difference in bFGF expression between the two groups.5.Immunohistochemical staining:BrdU,Nestin,Tuj1,Ng2 and GFAP have a small amount of neural stem cell proliferation in rat brain tissue in injury group.Number of positive cells in injury +10 Hz rTMS+stem cells group was evidently increased compared with injury group.The number in injury+0.5 Hz rTMS,injury+10 Hz rTMS and injury+stem cells group was between them.Moreover,Number of positive cells in injury+0.5 Hz rTMS+stem cells group was much higher than injury+0.5 Hz rTMS group.Number in injury+0.5 Hz rTMS+stem cells group was higher than injury+ 10 Hz rTMS and injury+stem cells group.6.BDA anterograde tracing:The pyramidal cells and axons of the cerebral cortex in the BDA injection area in injury group showed a small amount of fluorescence.The pyramidal cells of the cerebral cortex and the axon of the cerebral cortex in the injury+10 Hz rTMS+stem cells group were extensively fluorescent.Fluorescence in injury+0.5 Hz rTMS,injury+10 Hz rTMS,injury+stem cells,injury+0.5 Hz rTMS+stem cells and injury+10 Hz rTMS+stem cells group was between them.Conclusion:1.Repetitive transcranial magnetic stimulation combined with human umbilical cord blood mesenchymal stem cell transplantation can promote the proliferation of endogenous neural stem cells in rats with spinal cord injury,and high frequency is better than low frequency.2.Repetitive transcranial magnetic stimulation combined with transplantation of human umbilical cord blood mesenchymal stem cells can effectively improve the motor ability of rats with spinal cord injury,and the therapeutic effect was positively correlated with time.3.The high-frequency transcranial magnetic stimulation combined with human umbilical cord blood mesenchymal stem cell transplantation was more effective than low-frequency transcranial magnetic stimulation combined with human umbilical cord blood mesenchymal stem cells in rats with spinal cord injury,and significantly better than only use transcranial magnetic stimulation.4.Repetitive transcranial magnetic stimulation combined with human umbilical cord blood mesenchymal stem cell transplantation can improve neurological function,presumably by promoting the proliferation and differentiation of endogenous neural stem cells to complete. |
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