Proliferation and differentiation of mesenchymal stem cells modified with glial cell line-derived neurotrophic factor / 中国组织工程研究

ABSTRACT

BACKGROUND:Exogenous neurotrophic factors or chemical induction can induce rat bone marrow mesenchymal stem cells to differentiate into neuron-like cells. However, exogenous inductors exert a short inducible action, and their chemical substances inevitably have a negative impact on cellviability to limit the application prospects of bone marrow mesenchymal stem cells to a certain extent. OBJECTIVE:To investigate the effect of glial cellline-derived neurotrophic factor, green fluorescent protein gene transfection by adenovirus vector on biological characteristics of rat bone marrow mesenchymal stem cells, to observe the expression of glial cellline-derived neurotrophic factor and green fluorescent protein and the role of nutrition on bone marrow mesenchymal stem cells, and to explore the ability to differentiate into neuron-like cells induced by glial cellline-derived neurotrophic factor. METHODS:The bone marrow mesenchymal stem cells at passage 3 were transfected by recombinant adenovirus (Multiplicity of infection=10, 50, 80, 100, 150, 200). The experiment had two groups according to target genesbone marrow mesenchymal stem cells were transfected by Ad-GDNF-GFP in transfection group, and bone marrow mesenchymal stem cells were not transfected in control group. The expression of green fluorescent protein was detected by inverted fluorescence microscope. Transfection efficiency was calculated by flow cytometry. cells viability and the morphological changes of cells were compared respectively by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and inverted fluorescence microscope between the two groups. On days 5 and 10 after transfection, the expression of glial cel-derived neurotrophic factor mRNA was detected by PCR. On day 5, the expression of neuron-specific enolase was determined by immunofluorescence examination. On day 10, the expression of microtubule-associated protein 2 was identified. RESULTS AND CONCLUSION:By the end of 12 hours after transfection, the green fluorescent protein expressed in cells, and the fluorescence intensity gradual y increased with time. When the multiplicity of infection was 100, the fluorescence intensity was strong and stable, and the transfection rate was nealy 90%on day 3 after transfection. cellviability in the transfection group was strengthened after transfection. On day 5 after transfection, bone marrow mesenchymal stem cells expressed neuron-specific enolase, and neuron-like protrusions gradual y extended. On day 10 after transfection, bone marrow mesenchymal stem cells expressed microtubule-associated protein 2 and glial cellline-derived neurotrophic factor mRNA, and exhibited neuron-like morphology and interconnected synpases. The recombinant adenovirus, Ad-GDNF-GFP, can highly transfect bone marrow mesenchymal stem cells when the multiplicity of infection is 100, and glial cellline-derived neurotrophic factor can promote the proliferation of bone marrow mesenchymal stem cells and induce bone marrow mesenchymal stem cells to differentiate into neuron-like cells.