Experimental Study Of The Influence Of PBLAST49-mVEGF Gene Transfecation On Bone Fracture Healing

【Objective】Vascular endothelial growth factor can promise the generation of new blood vessels, which is very important in bone forming and bone fracture healing. Cytokin degenerated quickly in vivo, it is not convient to use continuously. By gene transfection, the extra gene DNA can be induced into host cell and express continuously and stably. The objective of this experiment include: (1) To learn the effect of extra VEGF gene expression on the proliferation and maturation of osteoblast cell. (2) To find an efficient way of extra VEGF gene transfer in vivo and in vitro. (3) To learn the influence of extra VEGF gene expression on bone fracture healing. 【Method 】(1) Enlarging, purification and identification of pBLAST49-mVEGF plasmid through E.Coli bacteria and RPM?4G plasmid purification kit. (2) Inducing the extra VEGF gene into osteoblast cell in vitro by lipofectamine, observe the expression of extra VEGF gene, and it's effect on osteoblast cell proliferation curve,cell cycle,synthesis of I type collagen and secretion of osteocalcium. (3) Establish the femur bone fracture model of S-D murine, infraperiosteum inject the pBLAST49-mVEGF plasmid ,lipofectamine mixture on the fracture site, observe the gross sample, X-R film and HE staining, to know it's influence on bone fracture healing. 【Result】(1) The OD260/OD280 of the purified pBLAST49-mVEGF plasmid is 1.81, and it's concentration is 7.4μg/μl. (2) After culture 1-2 days, the cell number of pBLAST49-mVEGF gene transfer group is larger than the controlled group, no statistical difference existed (p>0.05), however the difference between these two groups become significant since the 3rd day (p<0.05). (3) The double proliferation time of pBLAST49-mVEGF gene transfer group is 3 days, while the controlled group's time is 5 days. (4) The (G2/M+S)% of the 1-5 generation osteoblast cell in pBLAST49-mVEGF gene transfer group is higher than the controlled group, significant statistical difference existed (p<0.05). (5) The concentration of osteocalcium of the 1-5 generation osteoblast cell in pBLAST49-mVEGF gene transfer group is higher than the controlled group, significant statistical difference existed (p<0.05). (6) The expression of I type collagen of the 1-5 generation osteoblast cell in pBLAST49-mVEGF gene transfer group is stronger than the controlled group, significant statistical difference existed (p<0.05). (7) The bone fractur healing time of the pBLAST49-mVEGF gene transfer group is 2 weeks shorter than the controlled group. 【Conclusion】(1) Purified and much of pBLAST49-mVEGF plasmid can be obtained by E.Coli bacteria and RPM?4G plasmid purification kit. (2) Lipofectamine transfection is an efficient way for extra VEGF gene transfer in vivo and in vitro. (3) pBLAST49-mVEGF gene transfer can improve the proliferation, synthesis I type collagen and secretion osteocalcium. (4) pBLAST49-mVEGF gene transfer can facilitate the bone fracture healing.