| It is a tough problem to treat bone defect and nonunion caused trauma andpathology factor. It is the best way for researching a approach of promoting bonehealing to treat bone defect and nonunion. All kinds of factors of enhancingbone healing have been investigate, in numerous bone growth factor, bonemorphogenetic protein is one of the most important, it is the best osteoinductivefactor that has been thought now, its future is the best, Include bonemorphogenetic protein-2 (BMP2), bone morphogenetic protein -4 (BMP4) andbone morphogenetic protein-7 (BMP7). Bone morphogenetic protein (BMP) hasbeen apply in various kinds of animal model to induce bone formation andrepair bone defect. The animal experiments of enhancing bone healing by bonemorphogenetic protein (BMP) have been reported in literature. The predominantcharacteristic of bone morphogenetic protein (BMP) is to induce bone formationof allotopia and normal position, formation of cartilage and connective tissuewith bone tissue, like muscle tendon and ligamentous. The development andrebuildment and healing of bone tissue concomitance angiogenesis, neogenesisof blood vessel and bone is a juncture of disease'course reverse, neogenesis ofbone accompany angiogenesis. Angiogenesis correlate formation of nonagebone. Vascular endothelial growth factor (VEGF) is the best vigorousangiogenesis factor, Vascular endothelial growth factor (VEGF) specific activateendothelial cell, promote endotheliocyte proliferation and angiogenesis.Vascular endothelial growth factor (VEGF)'cartilage calcification pathway inbone formation process plays considerable role, Vascular endothelial growthfactor (VEGF) may produce a marked effect with humoral factor that regulateosteogenesis balance in bone formation. The intimate relation of Vascularendothelial growth factor (VEGF) and bone formation has been verified throughrivalry Vascular endothelial growth factor (VEGF), however the effect ofVascular endothelial growth factor (VEGF) isn't clear in bone formation. Withrespect to Vascular endothelial growth factor (VEGF) is or isn't requisition inbone formation induced bone morphogenetic protein (BMP), enhancingangiogenesis by application of VEGF gene is or isn't to enhance bone healing,have not been reported, the correlation of bone morphogenetic protein (BMP)and vascular endothelial growth factor (VEGF) has not been reported in boneformation, if ectogenic vascular endothelial growth factor (VEGF) increasesbone formation,if influences other important link of bone formation and bonerepair, is not clear now. Gene therapy research is one of the most future'sdomain, so it have been focal point in the last few years. But the preparation oftraditionary bone morphogenetic protein (BMP) proteinum and vascularendothelial growth factor (VEGF) proteinum have procedure complicated andoutput low and activity instability;local dose of utility and safe isn'tinaccessible;Gene therapy research have been focal point in the last few years.We apply technology line of EX VIVO, lipidosome is carrier in vitro, The ratbone mesenchymal stem cell cotransfected BMP2 gene and VEGF-165 genecompound with carrier stuff were inserted in muscle gap, to observe correlationof BMP2 gene and VEGF-165 gene in bone formation, if synergistic was present,we expect to discover original pathway to treat bone defect and bone nonunion.Part I: Construction of recombinant plasmidsRecombinant plasmids pIRES-VEGF165, pIRES-BMP-2 and pIRES-hBMP2-VEGF165 were constructed successfully with gene recombinanttechnique after identification with restriction enzyme.Part II: The culture and the proliferation and differention of bonemesenchymal stem cellsThe bone mesenchymal stem cells (BMSC) were obtained aspirate fromfemoral bone and humeral bone of rat. The adherent BMSC gave rise to coloniesand grew quickly under the microscope, some colonies and cells locating in thecenter of colonies expressed significant ALP (+), then mineralized nodule weredetected. The BMSC were obtained easily and the BMSC has high capability toproliferate and differentiate in vitro.Part III: Expression and detection of BMP-2 and VEGF-165 insupermate of cells culture after bone mesenchyal stem cells transfectedBMP-2 and VEGF-165Double gene coexpression plasmid were transferred into bone mesenchyalstem cells by liposome in vitro, content of BMP2 and VEGF-165 in supermate ofcells culture were drtected by enzyme linked immunosorbent assay, the resultsshowed that content of BMP-2 and VEGF-165 were gradually enhance along withhorary extention, bone mesenchyal stem cells transfected BMP-2 and VEGF-165were valid.Part IV: The research of BMP-2 and VEGF-165 double and a genemediated by liposome osteogenesis in rat bodyThe bone mesenchyal stem cells transfected BMP2 and VEGF-165 withgelfoam were transplanted into a muscle bag of the quadricept musle in rats.Animals were randomly divided into 2 groups, the first subgroup weredivided into 5 groups, which were transplanted with different kinds of graft.Animals in the first subgroup were killed 7, 14 and 28 days post transplantation,HE staining, microscope examination of the slices were carried out to assessedbone formation. The second subgroup were divided into 5 groups, which weretransplanted with different kinds of graft. Animals in the second subgroup werekilled 3 and 6 week post transplantation, examination of radioactive ray werecaaried out at 3 and 6 week to observe ectopic ossification.The results showed: VEGF may enhance and increase bone formation andbone healing, in conditions of VEGF-165 exist alone, VEGF-165 can induceangiogenesis and not induce bone formation, VEGF-165 can suction moremesenchyal stem cell.The radioactive film showed: BMP2 can induce ectopic ossification and isan important factor of inducing ectopic ossification. In the course of ectopicossification induced BMP2, VEGF-165 can enhance and increase ectopicossification and can not induce bone increase alone. Although VEGF is animportant factor in bone formation, it can not induce bone formation alone.
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