Experimental Studys On Mesenchymal Stem Cells Transfected With Vascular Endothelial Growth Factor Gene

[Objective]: In order to construct a tissue engineering artificial skull which has more blood supply and higher livability, we plan to use gene therapy method. For this reason, this experimental study has three purposes: (1) Enlargement, purification and identification of the pcDNA3-VEGF165 recombined plasmid is prepared for the gene transfection. (2) By studying of the growth characteristics of Mesenchymal Stem Cells and purification method, we can lay good foundation for the cultivation of MSCs in vitro. (3) To conform the validity that MSCs can transcript and secrete VEGF. [Method]: 1. Enlargement, purification and identification of the pcDNA3-VEGF165 recombined plasmid. 2. Primary and subcultured MSCs are cultivated in DMEM-LG with 10% fatal bovine serum(FBS) after gradient centrifugation in Peroll. 3. (1) pcDNA3-VEGF165 recombined plasmid is transfected into MSCs by Lipofectamine. (3) Identify the VEGFmRNA and VEGF by using RT-PCR and Western Blot method. [Result]: 1. (1) Three strips come to appear in the position of 6.0kb, 5.4kb and 600bp after gelose electrophoresis and their size accord with recombined plasmid DNA, pcDNA3 plasmid and VEGF165 gene accordingly. (2) Pure degree of the pcDNA3-VEGF165 recombined plasmid is: OD260/OD280=1.87. Its...