| BackroundsSpinal cord injury(SCI) is one of the most difficult problems of the medical profession in the world.SCI cause the interruption of connection between the brain and the part of the body under the injury level,including the sensory,movement, sphincter function impaired permanently.There is no effective treatment to it up to now.In the old opinion,the regeneration and functional recovery of the central nervous system after injury was absolutely impossible.Since 1981 the neuroscientist confirmed that the repair of the central nervous system structure after injury was possible,the study on repair of SCI has been becoming hot spot in many acdemic institute.The reasons that interfering axon regeneration after SCI were extremely complicated,First,primary and secondary injury on spinal cord caused a great of spinal cord functional neurons apoptosis and necrosis,which making the regeneration of axons difficulty.Second,a great quantity secretion and release of myelin-associated inhibitor and axon growth inhibition protein after SCI.The secondary glial scar impeded the axons growth and properly connected.Third,the decreased secretion of many neurotrophic factors resulting from partial cells apoptosis destroied the microenvironment benefiting for neurons repair and axon regeneration.The mechanism of SCI recovery is very complicated.SCI priminged various inhibit genes and regulate genes which interfering the regeneration of the spinal cord.Thus,there was rarely effection when aiming at single factor obstracting spinal cord regeneration.The measures of repairing SCI currently are mainly the following aspects:drug treatment,cell transplantation,tissue transplantation,improve the local microenvironment and union treatment.The traditional high-dose corticosteroids(methylprednisolone) impacting treatment could effectively reduce the secondary inflammation after SCI,and protect the residual damaging neurons and prevent the further apoptosis.At present,the studies claimed that lithium chloride(LiCl) and the immunosuppressant FKS06,and so on,could protect neurons from injury and could effectively promote axon regeneration.Cell transplantation is currently a hot research,the principle is to use some seed cells secreting a variety of neurotrophic,adhesion agents,nutrite and protecte the neuron after injury,and then induced axon regeneration and re-myelinization to repair SCI.Previous studies have provided a more mature seeds cells for SCI,such as:embryonic stem cells(ESCs), schwann cells(SCs),olfactory ensheathing cells(OECs),neural stem cells(NSCs), bone marrow mesenchymal stem cells(BMSCs),the umbilical cord blood stem cells (UCBCs).A large number of studies have confirmed that cell transplantation facilitates the restoration of spinal cord function.With the tissue engineering rapid development,a more advanced concept of SCI repairment appeared gradually that repair of SCI must include the organization levels reconstruction.Integrating effective treatments of SCI repair strategy,we could reduce glial scar formation at the same time induce the directional axon regeneration.At present,there were two engineering scaffolds applicating in spinal cord tissue,one was biodegradable polymer synthetic biological material,such as:polylactic acid,and polyglycolic acid copolymer,another was autologous or allogeneic tissue transplantation such as allogeneic embryonic spinal cord tissue,autologous muscle fiber frame,free of peripheral nerve(FPN),or the free vascularized peripheral nerve(VPN) tissue transplantation to repair spinal cord injury,and achieved a certain degree of effect. Another key factor for axon regenerate obstacle after SCI is the appropriate axonal regeneration micro-environment has been damaged,and myelin-derived protein (myelin,MAG,Nogo-A,Omgp) which inhibited the axon regeneration were greatly secreted,and lacking of various neurotrophic factor(BDNF and GDNF,NGF,NT3) and the glial scar formation hindered axon regeneration.To this end,inhibiting adverse inhibitory factor(IN-1 antibody),improving nutrition favorable factor (BDNF and GDNF) and improving local microenvironment became the research priorities.Recently,a new therapy strategy combining with several methods in different mechanism was used,which may have a better synergy to promote nerve repair after SCI.For example,compounding seed cells to synthetic polymer materials or peripheral nerve tissue which was the material of the stent transplanted into injuried spinal cord.Seed cells tansfected varies neurotrophic factor genes by gene engineering could secret specific nutritional factors to repair SCI,which have received satisfactory results.Accordingly,this study was designed to applicate GDNF gene-modified OECs transplantation in combination with antibody IN-1 which continuously injected by micro-pump to repair SCI.From this study,the nerve regeneration and function recovery of SCI animal will be investigated,at the same time,the possible mechanism of recovery in SCI will be discussed. Objectives1.To observe the effection of the neural stem cells differentiated to neurons by co-culturing of olfactory ensheathing cells(OECs) and neural stem cells(NSC),and provide theoretical evidences for cells union transplantation after spinal cord injury.2.To construct the model of spinal cord transection in rats,study the difference of model and the hindlimb functional recovery score after spinal cord injury under different gender.3.To construct the lentiviral vector with the GDNF gene and observe whether OECs be transfected effectivly by lentiviral vector with the GDNF in vitro.To observe the infected OECs whether long-term stably express GDNF through western blot.4.To study the repair effection to SCI with transplanting OECs modified with GDNF gene and jointing the injection of axonal growth inhibition antibody IN-1 in vivo.Methods1.OECs and NSCs were isolated from the 3-month-old SD rats' olfactory bulb and the hippocampus of neonatal rat respectively,cultured and identified in vitro. Purified OECs induce differentiation of NSCs through co-culturing with NSCs.2.Constructing spinal cord injury transsection model(T10) and observing the hindlimb spontaneous function recovery features after injury,comparing the differences of spontaneous functional recovery of the spinal cord transsection models under different gender.3.Using gene cloning and molecular biology methods to build a attenuated HIV lentiviral vector accompany with GDNF gene,and transfected olfactory ensheathing cells(OECs) in vitro,using the immunoblotting(Westem-blot) after transfection to observe whether the OECs long-term and stable expression of GDNF.4.Fifty adult female SD rats were undergone the entire transverse thoracic spinal cord injury and injected IN-1 with micro-pump persistently in subarachnoid in the injuried spinal cord.Then,OECs modified with GDNF gene were transplanted in injuried region.Using immunohistochemical staining method to observe the expression and distribution of the neurofibras and the residue neurons.Using corticospinal tract(CST) and spinal cord anterograde/retrograde tracing to observe the regeneration and distribution of corticospinal tract and spinal nerve fiber.BBB score were used to evaluate hindlimb motor function recovery level and the treatment effect of spinal cord injury.Results1.The purity rate of OECs after purified cultured is 93%,natural differentiated rate of NSCs differentiated to neurons is about 4%,differentiated rate of NSCs co-cultured with OECs differentiated to neurons is about 23%.2.The postoperative mortality and spontaneous hindlimb function BBB score have no significant difference between male and female rats with T10 transaction.There are very close about hindlimb BBB score in all the time among all animals. Hindlimb BBB score of all animals reached about 7 to 8 postoperative 4th week,and at later time maintained at the level of 8.But the urinary system complications in female rats significantly less than male rats.The postoperative care on female rats is more facilitated than male rats.3.Attenuated HIV lentiviral vector with the GDNF gene can be effective transfecte olfactory ensheathing cells(OECs) in vitro,the transfection rate is about 67%,the infected OECs can long-term survival and stably express GDNF. 4.It could be observed that hoechst marked olfactory ensheathing cells survivaed and migrated in the spinal cord postoperative 8th week.Using FITC fluorescence labeling NF-200 positive nerve fibers,spinal cord stumps atrophied and no axon regeneration could be observed in IN-1 group and the control group;disorderly regenerated axon could be observed in spinal cord injury district,but none of continuited nerve fibers through injury district in GDNF-OECs group and OECs group;but GDNF-OECs+IN-1 group,there were lots of regenerated axon,some of nerve fibers continuited through injury district.Fluorogold(FG) retrograde tracing show that a small amount of pyramidal cells were labeled by FG in GDNF-OECs+ IN-1 group,and GDNF-OECs group occasionally was observed nerve pyramidal cells marked by FG,the remaining three groups were not apperence.There were significant differences of BBB score of hindlimb functional movement among all groups except between the control group and the IN-1 group.Conclusions1.Neural stem cells can be induced to differentiate to neurons by olfactory ensheathing cells.2.Postoperative spontaneous hindlimb function BBB score could reach about 8 score and maintained at the level of 8 score.Postoperative mortality and spontaneous rats hindlimb motor function recovery BBB scores have no relation to animal gender. The postoperative care on female rats is more facilitated than male rats,so it is a good choice that applicate female rats to model.3.Lentiviral vector with the GDNF gene could effectivly transfecte olfactory ensheathing cells(OECs) in vitro,and infected OECs could long-term stably express GDNF. 4.The measure of transplantation of OECs modified with GDNF in combination with axonal growth inhibitor antibody IN-1 have the capability of protecting the remaining neurons and promoting corticospinal tract nerve axon regeneration,and guiding axonal regeneration through injury district and improving hindlimb motor function recovery.
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