Expression Of Recombinant Coexpression Plasmid Of PIRES-hVEGF₁₂₁cDNA/hBMP-4 In Rat Bone Marrow Mesenchymal Stem Cells And Its Effects On Differentiation

Objective To observe the expression of recombinant coexpression plasmid of pIRES-hVEGF₁₂₁cDNA/hBMP-4 in Transfected rat bone marrow mesenchymal stem cells(MSCs) and detect the differentiation of rat MSCs after transfection, which may provide a good basis for combination gene therapy of bone defects.Methods MSCs were isolated and cultivated from the bone marrow of SD rats by investigated their adherence-dependent growth characters. MSCs were analyzed by the flow cytometry to detect the surface antigens. Recombinant coexpression plasmids of pIRES-hVEGF₁₂₁cDNA/hBMP-4 were confirmed by restriction enzymolysis and then transfected rat MSCs by lipofectamine. Then the total RNA and total proteins extracted from the resulting combination were detected by RT-PCR and Western blot. Alizarin red staining detect the differentiation of rat MSCs after transfection.Result Flow cytometry analysis showed that MSCs were strong positive for CD90 but did not express CD45. Recombinant coexpression plasmid of pIRES-hVEGF₁₂₁cDNA/ hBMP-4 was transfected successfully and the plasmid could coexpress hVEGF₁₂₁cDNA and hBMP-4 efficiently in rat MSCs. Alizarin red staining presented strong positive for 14 days after transfectionConclusion Human VEGF₁₂₁ and BMP-4 gene can be successfully coexpressed in rat MSCs and induce rat MSCs to differentiate into osteoblast, which may offer the possibility of a novel approach of combination gene therapy of bone defects.