| BackgroundCharacteristics of spinal cord injury,especially acute spinal cord injury included sudden occurring,difficult treatment and poor prognosis in clinic,and rate of spinal cord injury incidence was high in home and abroad.With development of industrialization and urbanization procession,rate of spinal cord injury incidence increased highly at present.Although modern medicine made progress and cured many kinds of diseases in medical field,The treatment of spinal cord injury was still very difficult.There were no effective intervention of both prevention and treatment.It was far away from completed cure.The pathological process of spinal cord injury was very complicated,and included many kinds of mechanism.In recent decades,many experimental researches have been done,and have made great advancement especially in basic study.Which purpose is to find specific mechanism of spinal cord injury.disease will be cured completely in clinic according to different kinds of mechanism.The regeneration of spinal cord injury was the focus of biomedical research,which was also the difficulty that clinical doctors survived.The primary and secondary injury was main problems of repair after spinal cord was injured.Firstly,it was difficult to replace the degeneration and necrosis of neurons,and to restore original neurons.Secondly,it was also difficult to prevent and suppress the formation of scar in damaged region,which provided perfect micro-environment for neural regeneration.In the recent twenty years,great progress had been made in the repair and regeneration of spinal cord injury with efforts of many researchers.At present,the aid of most researches was to find seed cells for transplantation,which has been tried to be used in clinical treatment.Olfactory ensheathing cells could go through the peripheral nerve and the central nerve,promote axonal regeneration and remyelination of neurons,characteristics of olfactory ensheathing cells possed that of both astrocytes and Schwann cells.Olfactory ensheathing cells were confirmed to repair spinal cord injury at a certain degree in many experimental studies and trial of clinic treatment,which was one of the most important seed cells being investigated at present.Olfactory ensheathing cells were the best cell for transplantation currently,which was related to its unique biological properties closely.Olfactory ensheathing cells secreted and expressed brain-derived nerve growth factor,bone morphogenetic protein,neural cell adhesion molecule,laminin,platelet derived growth factor and low affinity nerve growth factor receptor.Olfactory ensheathing cells improved internal micro-environment in damaged area of spine cord,and repaired spine cord injury,inhibited harmful factors formation,reduced local inflammatory factors discharge.At the same time,olfactory ensheathing cells started myelinization and promoted axonal regeneration,re-established neural connection,and recovered function.In addition,olfactory ensheathing cells also inhibited proliferation of glial cells,reduced scar formation in spinal cord damaged area,and prevented void formation,which provided basis of repair and reconstruction of spine cord and facilitated new axons to go through damaged area.Nerve conduction and function were restored.Prognosis of spinal cord injury was closely related to effect of rehabilitation treatment and rehabilitation of patients,early rehabilitation intervention was an important kind of means to improve function for patients of spinal cord injury.Reasonable measures of physical therapy could significantly improve spinal function recovery for patients.Physical therapy was an important kind of treatment method to improve level of clinical effect,and were confirmed experimentally.Their treatment effect and clinical use of physical therapy were not identical according to different types,the common effect of physical therapy was to speed up the local blood circulation,improve local conditions,reduce the release of inflammatory factors,alleviate edema,reduce inflammation,and relieve pain effect.In addition,each kind of physical therapy also played its unique role.For example;it caused muscle contraction,humoral ionization and polarization,and rised local temperature higher.Electrical stimulation,one kind of physical therapy,improved micro-circulation,promoted the expression of neurotrophic factors and cell survival after transplantation.Through electric stimulation therapy,secondary damage of spinal cord injury was relieved after the initial injury,in late,complications of spinal cord injury were reduced,rates of survival and life quality of patient were improved,the clinical mortality could decreased obviously.Physical therapy for spinal cord injury was one of measures taken firstly in rehabilitation medicine for prevention and treatment of secondary injury,it improved local micro-environment,accelerated repair of neurons,and promoted function recovery of central nervous system through electrical therapy and electrical stimulation.The repair mechanism of spinal cord injury was complicated,intervention of repair also involved many methods,a single method for treatment was not the best choice,treatments combined with appropriate other kinds of measures were more necessary for repair of spinal cord injury.therefore the experiment was tried with application of olfactory ensheathing cell transplantation combined with physical therapy for the repair of spinal cord injury.Objective1.To prepare animal model of spinal cord injury.2.To choose suitable olfactory ensheathing cells used for transplantation in body.3.To study mechanism of physical therapy for repair,and find out interaction between physical treatment and cell transplantation.4.To investigate effect of repair by means of olfactory ensheathing cells transplantation.5.To explore effect of repair by means of olfactory ensheathing cells transplantation assisted with physical therapy,and observe interaction between olfactory ensheathing cells transplantation in body and physical therapy.Methods1.the rat spinal cord was completely cut off with iris knife,and the animal model of complete spinal cord injury was made at the level of 10 parts in the thoracic.2.olfactory ensheathing cells were purified by modified Nash differential adherence method and cytarabine chemical inhibition before transplantation.3.olfactory ensheathing cells were identified by p75 immunofluorescence staining under the fluorescence microscope.4.The adult S-D rats were randomly divided into four groups,the olfactory ensheathing cell transplantation combined with physical therapy group,the physical therapy group,the olfactory ensheathing cells group and the control group.5.From the second day the animal model was stimulated low frequency twice,each time for 15 minutes every day,and to observe the effect of physical therapy.6.the injured spinal cord and the distal end were injected into cells respectively.The improvement of the spinal cord function after transplantation was observed.7.the degree of repair of spinal cord injury was evaluated by BBB score,H-E staining,silver staining,horseradish peroxidase staining,somatosensory evoked potential,immunofluorescence staining,Western blot and Tunel staining.Results1.the model of spinal cord transection not only could ensure the survival rate of the experimental animals,but also achieve the effect of clinical simulation,which could meet the standard of complete spinal cord transection,and be used as an animal model of spinal cord injury2.the purity of cells by Nash differential adhesion method and cytarabine chemical inhibition was up to95%,which meet the requirement of cell transplantation.3.Olfactory ensheathing cells were positive of p75 immunofluorescence,and the biological characteristics did not change.4.BBB function scores according to groups the score was highest in olfactory ensheathing cells combined with physical therapy group,followed by the rest of the olfactory ensheathing celsl transplantation group,physical treatment group,control group,each group scores were statistically significant(P<0.05).H-E staining showed nerve fiber of spinal cord in the olfactory ensheathing cells combined with physical therapy group was more clear compared with the rest groups,there was directional scattering distribution of the hole,the other groups of spinal cord structure were unclear,and nerve fiber were twisted,there were a lot of syringomyelia addicted to the damaged region.Silver staining results showed that the number of nerve fibers was more in olfactory ensheathing cells combined with physical therapy group compared with other groups(P<0.05).the number of nerve fibers in the control group was few,Nerve fibers were cut off.Horseradish peroxidase staining showed that the number of neurons in the ensheathing cells combined with physical therapy group were most than other groups,results were statistically significant(P<0.05),olfactory ensheathing cells group,physical therapy group and control group were also statistically significant(P<0.05).It showed that each animal at 1 weeks after transplantation was detected wave forms of olfactory ensheathing cells combined with physical therapy group.2 weeks after transplantation,latency period was no differences between each group.In 4 weeks after transplantation,OECs combined with physical therapy group had the shortest latency period compared with other groups,the difference was statistically significant(P<0.05)in 6 weeks after cell transplantation,the latency in olfactory ensheathing cells combined with physical therapy group,olfactory ensheathing cell transplantation group,the physical therapy group and control group was statistically significance(P<0.05).Each animal began to curve in 1 week after cell transplantation,It could be detected in amplitude at 2 weeks after transplantation,all amplitudes were smaller with no differences between each group.At 4 weeks after transplantation amplitude waves were highest in olfactory ensheathing cells combined with physical therapy group animal,the difference was statistically significant(P<0.05),in 6 and 8 weeks after transplantation,volatility rised continually,the order from high to low was olfactory ensheathing cells combined with physical therapy group,olfactory ensheathing cell transplantation group,the physical therapy group and control group,the difference was statistically significant.(P<0.05).5.Immunofluorescence staining showed that olfactory ensheathing cells were found in vivo in olfactory ensheathing cells combined with physical therapy group and olfactory ensheathing cells transplantation group,but cell numbers were more in the former,the difference was statistically significant.(P<0.05).Western-blot analysis showed that Bax,Bcl-2,Caspase-3,Caspase-9 protein expression changed.in the olfactory ensheathing cells combined with physical therapy group Bcl-2 protein increased significantly(P<0.05),the of caspase-9 and caspase-3 expression reduced significantly(P<0.05),while Bax expression had no change.Tunel staining showed that numbers of olfactory ensheathing cells apoptosis in olfactory ensheathing cells combined with physical treatment group were less than the olfactory ensheathing cell transplantation group.the difference was statistically significant(P<0.05).Conclusions1.The animal model of complete spinal cord injury was simple,reproducible and reliable.It was suitable for animal experiment of spinal cord injury2.Olfactory ensheathing cells could be used to transfer into rats for spinal cord injury.3.The repair effect of olfactory ensheathing cell transplantation and physical therapy improved a certain extent,the best effect of repair was olfactory ensheathing cells combined with physical therapy,which improved highest than single method,but it was far from complete repair.4.The combination of physical therapy and olfactory ensheathing cells reduced the apoptosis of neurons and preserve the spinal cord function in the injured area. |
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