The Improvement Of Axonal Regeneration Microenvironment By NGFβ/HIF-1α Double Gene Transfected BMSCs

ObjectiveObserve the improvement of axonal regeneration microenvironment byNGFβ/HIF-1αdouble gene transfected bone marrow mesenchymal stemcells,to explore new methods for the treatment of SCI, to provide newperspectives and means for improving the axonal regenerationmicroenvironment.MethodsFirst, we construct lentiviral vector encoding NGF β and HIF-1α,anddetermine optimum MOI of lentivirus. Second, transfect BMSCs with measuredMOI, detect the expression of NGFβ and HIF-1αwith western blot. Next,construct co-culture system with transwell double plates which include neuronsand transfected BMSCs. Then culture the co-culture system in anaerobic tankfor48hours, detect the expression of NGFβ, HIF-1α in culture medium byELISA and measure the length of axons with Image pro-Plus software. Groups:Group A: neuron without BMSCs; Group B: neuron with BMSCs; Group C:neuron with NGF β transfected BMSCs; Group D: neuron with HIF-1αtransfected BMSCs; Group E: neuron with NGFβ/HIF-1αdouble transfectedBMSCs.ResultsThe result of BMSCs surface marker by flow cytometry is CD29positive, CD44positive，CD45negative, CD90positive, which corresponds to BMSCsimmune phenotype.The transfection efficiency of Le-RFP-NGF and Le-GFP-HIF-1α was84.83%and89.63%, respectively. Western blot analysis showed thattransfected BMSCs express target protein NGFβand HIF-1α.ELISA of the co-culture system showed that the NGFβexpression ofGroup C and Group E was significantly higher than Group A and Group B (P<0.05)，the HIF-1α expression of Group D and Group E was significantlyhigher than Group A and Group B (P <0.05). The experimental groups expressmore VEGF than control group.Measure the length of neuronal axons with Image pro-Plus，showed axonlength of Group E （NGFβ/HIF-1α double-transfected group）is greater thannon-transfected group (P <0.05),HIF-1α transfected group (P <0.05) andNGFβtransfected group (P <0.05).Conclusions1. Double-transfected BMSCs successfully expressed NGF β/HIF-1α,which creates a conducive microenvironment for neuronal axon growth.2. Neurons co-cultured with double-transfected BMSC can survive inanoxic environments well, thanks to the local microenvironment full of NGFβ,HIF-1α,important neuronal regeneration associated cytokines.