Transplantation Of PLGA Nerve Conduits With Neural Stem Cells Overexpressing Glia-derived Neurotrophic Factor Repairs Facial Nerve

PARTⅠConstruction of the lentiviral vector containing glia-derived neurotriphic factor and Virus packagingObjective:To construct the lentiviral vector containing enhanced green flurosecent protein and gliar cell line derived neurotriphic factor and provide well vector for gene therapy.Methods:Using PCR amplification,restriction endonuclease digestion,T4DNA ligase connections,GDNF will be inserted into the main virus vector pGC-FU,and constructed recombinant lentiviral vector(lenti-GDNF-EGFP).Lentivirus three plasmid system through Lipofectamine 2000 were transfected human embryonic kidney cell line-293 T,the titer of virus was examined.Result:We constructed the lentiviral vector expressed GDNF and EGFP gene and identified by sequencing and PCR successfully.The titer of the lentivirus is 2×10~8 TU / ml.Conclusion:we successfully constructed with EGFP and GDNF gene lentiviral vector which provide highly efficient and stable gene platform for gene therapy.PARTⅡExperiment study of neural stem cells culture and transfection and cultured PLGA conduits in vitroObjective:To construct a PLGA nerve conduit(NC)with NSCs overexpressing GDNF provides nutritional support for animal experiments.Methods:Using the embryonic rat hippocampal tissues,the primary NSCs producd and identified by Immunocytochemistry methods.The NSCs were transfected with lentivirus and the expression of EGFP was examined under fluorescent microscope. The transfection efficiency was examined by flow cytometry.The expression of GDNF was examined by Western blotting.The assay of GDNF after transfection was examined by ELISA test.The NSCs transplanted into PLGA nerve conduit after transfected,and observed under the field scanning electron microscopy.To identify the transplanted cells,we also transplanted the iron into the NSCs body through the Lipofectamine 2000 before the anmial experiment.Results:The construction of lentiviral vector(lenti-GDNF-EGFP)can be infected with NSCs and the transfection efficiency amount to 77.7 percent,the efficiency is still 73.7 percent after three months,Western blotting shew that GDNF expressed in the cell.ELISA test shew that the extracellular secretion of GDNF will be higher than the control groups,there were significant difference(P＜0.05).The transfected NSCs cultured PLGA conduits and produced the biological activity of artificial nerve conduit.Conclusion:GDNF gene can efficiently into NSCs through lentivirus mediated and constructed the bioactive artificial nerve conduit with PLGA tube.PartⅢTransplantation of PLGA nerve conduits with neural stem cells overexpressing GDNF repaires facial nerveObjectives:To transplant PLGA NCs with NSCs overexpressing GDNF repaires facial nerve.Methods:96 Sprague Dawley rats were subjected to right facial nerve transection, and NCs filled with transgenic NSCs were used to bridge the nerve gap(n=24). NCs containing NSCs,transgenic Schwann cells(SCs-GDNF),or empty NCs served as controls(n=24 per group).Facial nerve regeneration was assessed 2 to 12 weeks after surgery,through electrophysiological testing,immunohistochemical staining, and morphometric analysis of axons.The gene expression identified by the realtime PCR test in vivo.The transplanted cells were identified by iron staining and fluorescence outcome in vivo.Results:NSCs exhibited sustained GDNF expression after transplantation.Nerve action potential amplitude,myelin sheet thickness,and axonal area and number were significantly greater in the NSCs-GDNF group than the NSCs or empty NC group after 12weeks transplantation(p=0.000).Axonal area and number were also greater than the SCs-GDNF group(p=0.071).In vivo Iron staining and fluorescence results shew that the transplanted cells were foreign cells.Conclusion:Transgenic NSCs and PLGA NC transplantation have obvious advantages over repairing facial nerve and may improve regeneration of the facial nerve.