In Vitro Study Of Adipose-Derived Mesenchymal Stem Cells Transduced With Lentiviral Vector Carrying The Brain-Derived Neurotrophic Factor Gene

Brain-derived neurotrophic factor(BDNF)is a neurotrophic factor which plays an important role in neuronal survival and growth.Compared with other neurotrophic factors,BDNF has a broader receptor distribution,and BDNF can promote the release of other neurotrophic factors,enhancing the survival of nerve cells.It is difficult for BDNF protein to reach the lesion position after systemic administration for that it is a medium-sized protein.It has a short half-life so repeated treatment is needed.Gene therapy has aroused widespread concern,in which non-viral gene carrier is the research focus of disease treatment because of its high safety.Adipo1se-derived mesenchymal stem cells(ADSCs)are easy to be transfected with exogenous gene.ADSCs were considered to be a good gene carrier,which belongs to adult pluripotent stem cells.It has the advantages of abundant sources,easy extraction and no ethical problems.Gene therapy combined with stem cell therapy have synergistic effect on treatment.The aim of this study was to construct the BDNF-ADSCs to achieve the sustained expression of BDNF gene,and ADSCs could amplify and differentiate into the corresponding cell lines,which had a long-term mechanism.In this study,ADSCs were isolated and cultured from the abdominal fat of C57/BL mice,digesting by collagenase digestion.The purified cells were identified by cell surface markers,adipogenic and osteogenic differentiation ability,and their proliferation ability in vitro.The obtained cells were spindle-shaped,adhered to the vortex-like growth,and had osteogenic osteogenic differentiation ability.According to the results of flow cytometry analysis,it has a stem cell surface marker,rather than expressing hematopoietic,phagocytic and epithelial markers,consistent with mesenchymal stem cell phenotype.The ADSCs were transfected with recombinant lentivirus bearing BDNF gene,carrying enhanced green fluorescent protein(EGFP)gene and neomycin(Neo)gene for selecting stable expression clones..BDNF-ADSCs were obtained by fluorescence screening and G418 drug screening.The infection rate and transfection were analyzed by fluorescence microscopy.The osteogenic differentiation of ADSCs was treated by oil red O and alizarin red.The results showed that both alizarin red and oil red O staining showed positive results,and BDNF did not change its multiple differentiation potential.Colony forming units and growth curve determination was detected to analyze proliferation capacity of ADSCs and BDNF-ADSCs.Real-time PCR was used to detect the expression of cytokines and related pathways in ADSCs and BDNF-ADSCs.Its ability tomigrate and the protein expression was detected by Western-blot.The results showed that both could form colonies units,while BDNF-ADSCs had less colonies.Corresponding to the growth curve,it takes longer to grow at low cell density and has a relatively weak ability to proliferate.The transfection of BDNF gene did not change the stem cell characteristics of ADSCs,and its cell surface marker was the same as that before transfection.There were still adherent growth and multiple differentiation potentials,too.Through the growth curve,the transfected cells had long cell cycle,The expression of BDNF mRNA and correlated BDNF mRNA was increased by RT-PCR.The expression of BDNF m RNA was significantly higher than that of ADSCs.The expression of TRK-B,AKT-PIK3 and AKT-NFKb,which belong to its downstream pathways,were increased in different degree,the same with the related neurotrophic factors which BDNF couLd regulate in vitro.Western-blot analysis showed that NGF,BDNF protein expression can be successfully expressed in ADSCs in vitro and play a key role in promoting other neurotrophic factors.