Preliminary Studies Of Differentiation Into Neuron-like Cells And Protection To The Cochlear SGNs Of Bone-marrow Mesenchymal Stem Cells Transfected By Brain-derived Neurotrophic Factor Gene

PartⅠConstruction and expression of recombinant pcDNA3.1(-)-hBDNF plasmidObjective Neurotrophic factors(NTFs) have great influence on the growth,development and function-maintenance of nervous system. Recent years,some members of NTFs have been cloned and applied into the diseases of nervous system.Much progress has been made in these studies.Brain-derived neurotrophic factor(BDNF),as a member of the family of NTFs,is closely connected with mammalian peripherial auditory system.BDNF not only plays an important role in the regulation of development,maturity,prolifaction and differenation of auditory neurons, but also has strongly ability to remodel the specific function in adulthood and prevent or reduce secondary degeneration after the destruction of auditory neuron.On this condition,the purpose of our study is to make basis for gene engineering production of BDNF and make construction of eukaryotic expression vector:pcDNA3.1(-)-BDNF,and to detect the expression of BDNF after the vector transfects human embryo kidney 293 cells.The source of BDNF gene was provided for the luther study of the prevention and treatment of sensorineural deafness.Methods Human brain-derived neurotrophic factor gene(hBDNF) was obtained by RT-PCR method,and recombinant pcDNA3.1(-)-hBDNF plasmid was constructed by connecting the gene with pcDNA3.1(-) plasmid.The recombinant plasmid was analyzed and identified by the EcoRⅠand BamHⅠrestriction endonuclease after transformed,screened and amplified.Small amounts of recombinant plasmid was extracted and sequenced,then the correct recombinant pcDNA3.1(-)-BDNF plasmid was massively extracted with QIAGEN-TIP100 kit.HEK293 was transfected by pcDNA3.1(-)-BDNF plasmid,BDNF in the HEK293 was detected with Western Blot.Results RT-PCR amplification of the hBDNF gene was performed. Recombinant pcDNA3.1(-)-hBDNF plasmid was successfully constructed by connecting the hBDNF gene with pcDNA 3.1(-) plasmid.The human BDNF gene encoding mature peptide was confirmed to be cloned by identified with restriction enzymes and sequenced.BDNF in the HEK293 transfected by pcDNA3.1(-)-BDNF plasmid was confirmed with Western Blot.Conclusions The recombinant plasmid of pcDNA3.1(-)-hBDNF was constructed successfully,and it could be used in the further experiments for gene therapy. PartⅡThe observation of bone-marrow mesenchymal stem cells differentiate into neuron-like cells in vitro after transfected by brain- derived neurotrophic factor gene with electroporationObjective To investigate the differentiation of bone-marrow mesenchymal stem cells(BMSCs) into neuron-like cells in vitro after transfected by human Brain-derived neurotrophic factor(BDNF) gene.Methods Human BDNF genes were cloned and recombinant pcDNA3.1(-)-BDNF plasmids were constructed.Adherent cells were isolated and cultured from bone marrow of five guinea pigs.The morphology of these cells was observed by microscope and the surface antigen was detected by flowcytometry with CD34,CD44 and CD45 antibody.BDNF gene was transfected to BMSCs with electroporation,the transfected BMSCs were induced by ratinoic acid(RA) after bolted by Geneticin-418(G418),then the differentiated BMSCs were identified by immunocytochemistry of neuron-specificenolase(NSE),Nestin and glial fibrillary acid protien(GFAP)antibody.Results The culture cells had the typical morphology and surface antigen of BMSCs,the efficiency of human BDNF gene transfected BMSCs with electroporation was about 25%,the transfected cells were similar to nerve cells on appearance and could express NSE,Nestin and GFAP.Conclusions BMSCs transfected by BDNF gene can differentiate into neuron-like cells in vitro,electroporation can boost the transfection efficiency,RA has the effect to promote induction.PartⅢPreliminary observation of bone-marrow mesenchymal stem cells transplant into cochlea after transfected by brain-derived neurotrophic factor geneObjective To investigate the function of differentiation and protection of bone-marrow mesenchymal stem cells(BMSCs) in damaged cochlea after transfected by human brain-derived neurotrophic factor (BDNF) gene.Methods BMSCs were induced and marked after transfected by hBDNF gene in vitro.These BMSCs(BDNF- BMSCs) were transplanted into tympanic scala of the guinea pigs which were deafened by amikacin(AK).The control groups were designed in which artifical perilymphatic fluid(APF),BMSCs or BDNF gene was injected into cochlea alone.The cochlea was respectively obtained and in paraffin-embedded on the week 1,2 and 4 after injected,and cut in a paramodiolar plane subsequently.Histopathological changes were observed and analyzed by staining with HE,the density of SGNs was calculated,the BMSCs distribution was reviewed,differentiation of BMSCs in vivo and survival of SGNs was detected with immunohistochemistry of NSE,Nestin and GFAP antibody.Results Construction of the guinea pig cochlea was damaged when amikacin was injected into abdominal cavity,the number of SGNs was decreased.The result of immunochemistry analyzed with average optical density(AOD) showed:the AOD of the BDNF-BMSCs group in vivo was not obviously different from in vitro on 1W and 2W,but significantly reduced on 4W(P＜0.05),and was significantly higher than the BMSCs group on 1W,2W and 4W(P＜0.01)BMSCs which were differentiated or not could survive and scatter over some areas in the cochlea.Some induced BMSCs were positive for NSE,Nestin and GFAP,while only a few uninduced BMSCs were positive BMSCs,BDNF gene or BDNF- BMSCs could all rival the ototoxicity of amikacin in the cochlea,the protection taken by BDNF-BMSCs was the most obvious.In the BDNF- BMSCs group it could be confirmed about SGNs that the morphous changed smallest,the density and average optical density for NSE was the highest.The difference was significant compared with the control groups(P＜0.05 )Conclusions The differentiated ability of BMSCs induced by BDNF gene in vitro is invariable in about two weeks when they are transplanted into the cochlea,but reduces on 4W.The better protection of inner ear is confirmed when BDNF-BMSCs are transplanted into cochlea compared with transplanting BMSCs or BDNF gene alone.