Adenovirus-mediated Glial Cell-derived Neurotrophic Factor Gene Via Wnt Signal Pathway Inducing Bone Marrow Mesenchymal Stem Cells To Differentiate Into Neuron-like Cells

Objective To study the effect of adenovirus-mediated glial cell-derived neurotrophic factor gene on bone marrow mesenchymal stem cells differentiating into neuron-like cells,and explore the role of Wnt signal pathway in BMSCs differentiating into neuron-like cells.Methods BMSCs were isolated from SD rats.Surface makers of BMSCs were detected by flow cytometry.BMSCs at passage 3 were divided into three groups:the transfected group(transfected by AD-GDNF-GFP),the blocked group(transfected by AD-GDNF-GFP and DKK-1 to block the Wnt signaling pathway)and the negative control group(cultivated in DMEM with 10%fetal bovine serum).Expression of GFP in the transfected group was observed by inverted fluorescence microscope after transfection.The cell viability was evaluated by MTT.Expression of glial cell-derived neurotrophic factor was detected by western blot at different time.Expression of NSE,MAP-2 and β-Tublin Ⅲ were observed by immunofluorescence.RT-PCR was used to detect the expression of neurotrophic factor mRNA.After induction,immunofluorescence and western blot were used to detect the expression of β-catenin and RT-PCR used to detect the expression of Wnt1a、Wnt3a、Wnt7a mRNA to explore the role of Wnt signal pathway in BMSCs differentiating into neural cells.Results BMSCs at passage 3 were mostly spindle-shaped and positive for CD29、CD90 but negative for CD11b、CD34.The expression of GFP could be observed by inverted fluorescence microscope after 24 hours of transfection.With the extension of time,the fluorescence intensity of GFP was gradually enhanced.After 7 days of transfection,the expression intensity of green fluorescent protein GFP was the highest,and the fluorescence intensity was distinctly weakend after 14 days of transfection.The expression of GDNF could be detected by western blot until 14 days after transfection.Expression of NSE could be detected by immunofluorescence positively after 5 days and MAP-2 and β-Tublin Ⅲ could be detected after 10 days in the transfected group.Express ion of β-catenin and NSE could be detected in the meanwhile afte 5days of induction.The results detected by RT-PCR showed obvious up-regulation mRNA expression of Wnt1a、Wnt3a、Wnt7a in the transfected group.Conclusion Adenovirus-mediated glial cell-derived neurotrophic factor gene can induce bone marrow mesenchymal stem cell to differentiate into neuron-like cells.Wnt signal pathway plays an important role in Adenovirus-mediated glial cell-derived neurotrophic factor gene inducing bone marrow mesenchymal stem cell to differentiate into neuron-like cells.