Objective To detect the efficiency of the recombinant plasmids, pDsVEGF165 Red1-N1 and pIRES2-BMP2-EGFP, in mammalian cells and to provide the foundation of gene therapy in vivo. Methods 293-T cells grown in six-well plates at a density of 1×105 cells in 0.5 ml of DMEM per well were transfected with pDsVEGF165Red1-N1 and pIRES2-BMP2-EGFP plasmids DNA or pDsRed1-N1 and pIRES2-EGFP empty vector DNA by means of DOTAP in condition recommended by the manufacture（Roche）.Plasmid DNA (total 2.5 ug of two plasmids) was dissolved in 25 ml HBS and mixed with 15 ml DOTAP reagent dissolved in 50 ml HBS, and incubated at 15℃ for 15 min to allow DNA-DOTAP complexs to form. Then 0.5 ml medium without serum was added, and the DNA-DOTAP solution was applied onto a monolayer of 1×105 cells in six-well plates. The cells were incubated with the complexes for 6 h at 37℃ 5％CO2 incubator. Following incubation , 1 ml of growth medium containing 10% serum was added and removing the transfection mixture. 48 h later,DsRed and EGFP were observed by fluorescent microscope and confocal laser-scanning microscope,mRNA of genes were detected by RT-PCR and proteins expressed in cells were detected by Western Blotting. Results Recombinant plasmids pDsVEGF165Red1-N1 and pIRES2-BMP2- EGFP all allow proteins transient expression in mammalian cells, which was confirmed by fluorescent microscope,CLSM and Western Blotting. Conclusion Transfection of the mammalian cells with plasmids pDsVEGF165 Red1-N1 and pIRES2-BMP2-EGFP leads to transient expression VEGF165 or BMP2.
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Part 4 The function of recombinant plasmids pDsVEGF165Red1-N1、pIRES2- BMP2- EGFP in long bone defect of rabbits
Objective To discuss the function of recombinant plasmid, pDsVEGF 165Red1-N1、pIRES2-BMP2-EGFP, in neovascularication and bone formation of long bone defect of rabbits. Methods A total of 30 healthy adult rabbits were randomly divided into five groups. Right forelimb was operated as experimental limb.The first group: collagen membrane,n-HA/PA, pDsVEGF165Red1-N1 and pIRES2-BMP2-EGFP plasmids were applied; the second group: collagen membrane,n-HA/PA, pDsVEGF165Red1- N1 plasmids were applied; the third group: collagen membrane, n-HA/PA,pIRES2-BMP2- EGFP plasmids were applied; the fourth group: collagen membrane, n- HA/PA (no combined plasmid DNA) were applied; the fifth group: Constructing the long bone defect, no treatment. The samples were examined by gross, X-ray, histomorphology, SEM and ECT in 2W, 4W,8W. Results (1) The expression of fluorescent (EGFP or RFP) can be observed by fluorescent microscope in muscles around the bone defect at two, four, eight weeks; (2) At 2 weeks, no distinct difference among groups was observed. ECT showed that the blood flow of the first group in the bone defect site was increased(P<0.05)、、、、、、>
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