Research On Bone Mesenchymal Stem Cells Cytocompatibility With PLGA Biomaterial After Transfection Of PEGFP-N₂-VEGF Plasmid

Objective: Transfection of pEGFP-N2-VEGF Plasmid into mesenchymal stem cells and then research on its cytocompatibility with PLGA.Methods: 1, primary culture by density gradient centrifugation to obtain bone marrow mesenchymal stem cells, conventional change liquid, cryopreservation and recovery, flow cytometry check it's cell cycle; 2, containing pEGFP-N2-VEGF gene plasmid strains of E. coli bacteria shaking overnight, with Grand endotoxin plasmid extraction kit, using UV spectrophotometry and agarose gel electrophoresis detection of its concentration and purity; 3, liposome encapsulation method used to transfer pEGFP-N2-VEGF gene into bone marrow-derived mesenchymal stem cells, PCR detection of the effect of transfection, immunohistochemistry to observe whether there is protein expression; 4, transfection as usual against the cultivation model in PLGA on bone marrow-derived mesenchymal stem cells, respectively, using fluorescence microscopy and scanning electron microscope observation; 5, MTT (colorimetric MTT test) was used to detect pEGFP-N2-VEGF transfected cell viability before and after, divided into 4 groups:①bone marrow-derived mesenchymal stem cells group,②bone marrow-derived mesenchymal stem cells transfected with pEGFP-N2-VEGF group,③bone marrow-derived mesenchymal stem cells + PLGA group,④bone marrow-derived mesenchymal stem cells transfected with pEGFP-N2-VEGF group + PLGA group.Results: 1, primary bone marrow-derived mesenchymal stem cells were slender spindle, clone-like growth; many times after subculture, cells become flat. the second generation of flow cytometry of bone marrow mesenchymal stem cells more than 90% of the cells are in G0/G1 phase. And the sixth generation in G0/G1 phase accounts for about 80%. 2, UV-spectrophotometry after bacteria that shake the pEGFP-N2-VEGF plasmid solubility for 1380μg/ml, agarose gel electrophoresis shows that the corresponding bands; 3,pEGFP-N2-VEGF gene into bone marrow-derived mesenchymal stem cells, PT -PCR detection of the amplification characteristic bands appear, compared with DNAmark, as amended, in line with the location; 48h later, cellular immune staining, VEGF-positive antibody response. 4,it can be genetically modified in the fluorescence microscope, scanning electron microscopy observation of cells and better interaction between the material after transfection; 5, MTT before and after transfection show 4 group light wavelength absorption values at 492nm, respectively: 0.309±0.113 , 0.350±0.176,0.295±0.112,0.336±0.172; in the wavelengths 629nm were 0.104±0.036,0.121±0.050,0.085±0.035,0.102±0.053,①group and②group,③Group and④group statistically are not statistically significant (p>0.05).Conclusion: 1, density gradient centrifugation will be a good method to obtain bone marrow-derived mesenchymal stem cells of sufficient number strong differentiation potential; 2, bone marrow-derived mesenchymal stem cells transfected with pEGFP-N2-VEGF gene and protein could express in the molecular-level; 3, transfection pEGFP-N2-VEGF gene can be used as markers in bone marrow mesenchymal stem cells is an effective way for dynamic observation of cells and the interaction between the material,it lay a certain foundation; 4, transfected with pEGFP-N2-VEGF gene does not affect the bone marrow-derived mesenchymal stem cells and its ability with PLGA material,which provides a new means for vascular tissue engineering.