Study On Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Transfected With NT-3 Gene Into Neurons In Dendritic Amphiphilic Peptide Gel

Objective:Through three-dimensional culture of NT-3 gene-modified rat bone marrow mesenchymal stem cells into the resinous amphiphilic peptide gel,to study the effect of differentiation of rat bone marrow mesenchymal stem cells on neurons.Method:Isolated and extracted bone marrow mesenchymal stem cells from SD rats at 4-6 weeks under aseptic condition,and identified by flow cytometry and in vitro osteogenesis and adipogenic differentiation.Using a cytomegalovirus(CMV)as a promoter to construct an adenoviral vector carrying the NT-3 gene and the green fluorescent marker gene(GFP),AdvNT-3.Transfected the rat bone marrow mesenchymal stem cells with different multiplicity of infection(MOI value).Two days after transfection,the optimal viral multiplicity of infection(MOI)was determined based on the expression of green fluorescent protein under fluorescence microscope.The rat bone marrow stem cells transfected with the virus with the best MOI value are NT-3-BMSCs.Add 1 wt% peptide solution to DME/F12 solution with an equal volume density of 110^6/ml BMSC,to form a three-dimensional cell gel complex.And cells were distributed inside the gel to form a three-dimensional culture system.The experiment was divided into three groups.Group A: BMSC two-dimensional culture group.Group B: NT-3-BMSC two-dimensional culture group.Group C: Three-dimensional culture group of NT-3-BMSC with gel.After the three groups were cultured in vitro for seven days,used immunofluorescence and qPCR to detect the expression of three neuronal specific markers MAP-2 and β-III Tubulin.Result:Isolated cultured cells were identified as bone marrow stem cells by flow cytometry.Forty-eight hours after transfection of bone marrow mesenchymal stem cells by virus,green fluorescent expression was observed under fluorescent microscope.And with the increase of the MOI value,the fluorescence expression became stronger and peaked when MOI equaled to 100.When MOI equaled to 300,some cells were observed dead under microscope.Therefore,the optimal MOI value is 100.Peptide solution triggered by DMEM/F12 formed a three-dimensional gel,which was a visually transparent gel but appeared to be three-dimensional mesh fibers when observed under electron microscope.Immunofluorescence result showed negative expression of neuron-specific markers MAP-2 and β-III Tubulin in group A,and positive expression of neuronal specific markers MAP-2 and β-III Tubulin in group B and C,and the positive rate in group C was higher than that in group B.qRCR results are the same as those of immunofluorescence.The relative expression levels of neuronal specific markers MAP-2 and β-III Tubulin in group B and C were higher than those in group A.And the relative expression of MAP-2 and β-III Tubulin in group C was higher than that in group B.Therefore,NT-3 gene-modified BMSCs cultured in three-dimensional gels can promote the differentiation of cells into neurons,and three-dimensional culture is better than two-dimensional culture.