Transplantation Of Neurotrophin-3 Gene Modified Neural Stem Cells For Spinal Cord Injury Treatment In Rats

Objective:To investigate therapeutic effects of transplantation of Neurotrophin-3(NT-3) modified fetal spinal cord-derived neural stem cells (NSCs) for spinal cord injury(SCI) repairing.Methods:eukaryon expression vector pEGFPN1-NT-3 recombinant plasmid wasconstructed and transfected into NSCs to establish EGFP-labeled NT-3 gene modifiedNSCs via nucleofection.Therapeutic effects of NSCs transplantation at different times aftcrSCI were compared and most suitable time point for NSCs transplantation was determincd.After NT-3-NSCs transplanted into SCI rats,therapeutic effect of NT-3-NSCstransplantation was evaluated by behavioral tests,and analyses of the survival,differentiation and gene expressions of grafted cells at injured sites.Results:Behavioral tests presented the transplantation of NT-3 gene modified NSCscould make SCI rats get more behavioral recovery than the transplantation of unmodifiedNSCs.Immunohistochemical analyses demonstrated NT-3 modification could improvc thesurvival and differentiation into oligodendrocytes of grafted cells.Molecular biologicalexperiments revealed NT-3 modified NSCs could obtain stronger NT-3 and MBPexpression than unmodified NSCs after transplantation.Conclusion:Transplantation of NT-3 modified NSCs is a potential therapy for SCIrepairing,which yields better effect than transplantation of unmodified NSCs, transplantation of NT-3 modified NSCs has a brilliant future for SCI treatment.PART ONE:Construction of recombinant plasmidObjective:To construct NT-3 gene eukaryon expression vector pEGFPN1-NT-3recombinant plasmid.Methods:The coding sequence of NT-3 was amplified andconnected to pMD19-T Simple Vector;after identification,NT-3 gene and plasmid vectorpEGFPN1 were connected.Results:The result of double enzyme digestion of recombinantplasmids was consistent with expectation.Conclusion:NT-3 gene eukaryon expressionvector pEGFPN 1-NT-3 recombinant plasmid was successfully constructed.PART TWO:Culture of neural stem cellsObjective:To stably culture fetal spinal cord derived neural stem cells.Methods:Spinal cord tissue was obtained from fetal Sprague-Dawley rats at embryonic day 14,neural stem cells were cultured and their multipotentiality was detected.Results:Fetalspinal cord neural stem cells formed neurospheres and were Nestin immunoreactivc.Conclusion:Fetal spinal cord neural stem cells were successfully cultured and their stemcell multipotentiality was maintained.PART THREE:NT-3 gene modificationObjective:To construct EGFP-labeled NT-3 gene modified neural stem cells.Methods:After neural stem cells were transfected with recombinant plasmidpEGFPN1-NT-3 via nucleofection,the cells were identified via RT-PCR,Western blot and immunocytochemical assessments.Results:RT-PCR and Western blot results prcscntedstrong expression of NT-3 in transfected cells;immunocytochemical staining demonstratedNT-3 and EGFP immunoreactions in transfected ceils.Conclusion:EGFP-labeled NT-3gene modified neural stem cells were successfully constructed and could express NT-3 andEGFP.PART FOUR:Establishment of spinal cord hemisection modelsObjective:To establish thoracic 9 right-sided spinal cord hemisection rat models.Methods:Spinal cords of female Sprague--Dawley rats were right-sided hemisected atthoracic 9 level,behavioral observation and tests were administered postoperatively.Results:Right hindlimb paralyzed after operation;hemisected rats were scored were lessthan 2 in BBB test and fell frequently in grid test.Conclusion:Thoracic 9 right-sidedspinal cord hemisection rat models were successfully established.PART FIVE:Neural stem cells transplantationObjective:To determine the most suitable neural stem cells transplantation time afterspinal cord injury.Methods:The therapeutic effects of neural stem cells transplantation atdifferent times after spinal cord injury were compared to determine the best transplantationtime point.Results:The rats received neural stem cells transplantation at 3rd,7th and 14thdays after spinal cord injury presented better results than spinal injured rats,rats obtainedbest recovery when neural stem cells transplanted at 7th day after spinal cord injury.Conclusion:The most suitable neural stem cells transplantation time was around one week after spinal cord injury.PART SIX:NT-3 gene modified neural stem cells transplantationObjective:To study the behavioral recovery effect of NT-3 gene modified fetal spinalcord derived neural stem cells transplantation for spinal cord injury rats.Methods:Thebehavioral tests were applied to evaluate the behavioral recovery in spinal cord injury ratsafter transplantation of NT-3 modified neural stem cells;the therapeutic effects of NT-3modified and non-modified neural stem cells transplantation were compared.Results:Therats received NT-3 modified or non-modified neural stem cells transplantation after spinalcord injury presented better results than controls,rats obtained best recovery when receivedNT-3 modified neural stem cells transplantation.Conclusion:The behavioral recoveryeffect of neural stem cells transplantation could be improved by NT-3 gene modification inspinal cord injury rats.PART SEVEN:Detection of transplanted cellsObjective:To study the survival,differentiation and gene expression of transplantedNT-3 modified fetal spinal cord derived neural stem cells.Methods:The survival anddifferentiation of transplanted cells were detected via immunohistochemistries,relatedgenes expressions were detected via molecular biologies;which were compared withtransplantation of non-modified neural stem cells,Results:Immunohistochemical stainingpresented NT-3 modified neural stem cells got better survival,NT-3 secretion anddifferentiation into oligodendrocytes in injuried spinal cords;molecular biologicalassessment demonstrated NT-3 modification could increase NT-3 and MBP expression attranscription and protein levels.Conclusion:NT-3 gene modification could improve the survival,differentiation and related genes expression of transplanted neural stem cells inspinal cord injury rats.