| Bone defect caused by tumor, inflammation, trauma and heteroplasia are frequently encountered in clinical. Both bone regeneration and revascularization are needed in reparation of bone defect, and the revascularization of the bone play an important role in it. Autografts, allografts and artificial bone substitutes are general methods in reparation of bone defect. But all of these methods have there inherent problems and limitations. Autografts are limited by the lack of adequate supply and donor site morbidity. Allografts and artificial bone substitutes are not backed by adequate cellular seeds. Nowadays, the methods of tissue engineered bone have the method compounded substitutes and cellular seeds, substitutes and active factors, substitutes, cellular seeds, and active factors. Although the experiments with these methods have gained some effect, the results do not reach the ideal effect. In these experiments there are not a suitable. The freezing and drying bone still seems a fitting substitute among them.Nowadays, as a commodity, immunological rejection of freezing and drying bone has seldom seen and cell-based bong tissue engineering has emerged as a promising alternative for bone repair. So in our experiment, we applied with freezing and dryingbone and bone marrow stem cells (BMSC) as cellular seeds. In these conditions, freezing and drying bones provided fine interspaces structure and good adhesive and grow conditions. At the same time, BMSC which transfected vascular endothelial growth factors (VEGF) as cellular seeds provided the seeds and stimulating factors.1, Cultured rabbit BMSC in vitroObjective: To study on the influence on transformation of gene transfect BMSC and compare with normal BMSC by graphic the growth curve, we cultured and expended BMSC in vitro and observed the its histopathologic features. Methods: Isolated rabbit BMSC by adhesion method and cultured it at 37C , 5%CO2 incubator. Graphic the growth curve after observed the growth of primary cells and passage cells. Cultured BMSC in calcified solution and observe its potential of osteogenic. Results: The primary cells formed a monolayer in 18th day . Von-kossa staining presented mineral deposition after the cells had been cultured 30 days in calcified solution. Conclusion: BMSC can be isolated and cultured easily. It has a excellent speed of amplification and potential of osteogenic which can form mineralized nodules.2, Construction of VEGF retroviral expression vectorObjective: To construct a high effective retroviral expression vector of VEGF for the next experiment. Methods : Taken hVEGF165 cDNA from pT7Blue/ hVEGFies plasmid and inserted into polylinker site of pcDNAs to construct pcDNAs/ hVEGFies, packed with PA317, followed by the selection of G418.Results: The pcDNAs/ hVEGFies retroviral expression vector was successfully constructed and got the same result as GeneBank in the DNA sequencing analysis of hVEGFies. Conclusion: The pcDNAs/ hVEGFies retroviral expression vector was successfully constructed and laid a good foundation on continuing research.3, Expression and effect of VEGF in rabbit BMSCObjective: To detect the expression of objective gene and the effect of stimulate angiogenesis after vascular endothelial growth factor(VEGF) was transferred to rabbit mesenchymal stem cells(BMSc) mediated by retrovirus. This study laid the foundation for its further use in vascularized tissue engineered bone. Methods : A retroviral vector containing VEGF gene was constructed and it was expressed after it was transferred to BMSc. The protein expression was detected by immuohistochemistry and its effect of stimulating angiogenesis was observed by microvessal count (MVC). Results: The retroviral vector containing VEGF gene was constructed successfully. Immuohistochemistry showed that it expressed VEGF protein in BMSc plasma (brown staining) and the experimental study confirmed that its MVC was higher than that in control group (P<0.01). Conclusion: Gene transfer technology mediated by retroviral vector...
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