Construction Of BMP-2 Gene And IGF-1 Gene Co-expression Vector And Effect Of Transfected On Osteogenic Differentiation Of Rat Bone Mesenchymal Stem Cells

It is a tough problem to treat bone defect and nonunion caused trauma infection and tumor factor.It is the best way for researching a approach of promoting bone healing to treat bone defect and nonunion.People always treat bone defect by single gene,now it has cooperation and facilitative effect between growth factors through a long time study.Double genes can promote cell proliferation and differentiation.All kinds of factors of enhancing bone healing have been investigate,in numerous bone growth factor,bone morphogenetic protein is one of the most important,it is the best osteoinductive factor that has been thought now,its future is the best.TGF-β,PDGF,FGF and physical stimulation will regulate bone metabolic process,promote bone repair.Bone morphogenetic protein(BMP)is only a pro-morphous,induce undifferentiated mesenchymal cell to differentiate osteoblast,but can't promote cells proliferation.BMP has better educe its osteoblast differentiation,with other fractors.IGF-I is cells mitogen,can promote cell proliferation and differentiation.IGF-1 promots membrane bone formation and repairs bone defect.IGF-1 can regulate bone formation and cells copy.It reports that rhIGF-Ⅰand rhBMP-2 promote cell proliferation and differentiation. We hope that IGF-Ⅰand BMP-2 can promote cell proliferation and differentiation, if IGF-Ⅰand BMP-2 imported clles through transfection technique.In this study,to approach IGF-1 and BMP-2 correlation and role in the bone formation procedure,we can find a now method to elevate bone healing and repair bone defect.Vector containing combinations of condensed plasmid DNA encoding for IGF-1 and BMP-2,pEGFP-N1/IGF1-BMP2,were constructed by molecular cloning and gene transfectiont echnique.The cultured bone marrow mesenchymal stem cells(BMSCs)were transfected with cation liposome-mediated transfection method to express the exogenous IGF-1 and BMP-2.The transfected BMSCs could improve the bone regeneration and bone healing.It also provided modified seed cells for bone tissue engineering.We expect to discover original pathway to treat bone defect and bone nonunion.PartⅠ:Construction of recombinant plasmidsRecombinant plasmids pEGFP-N1/IGF1,pEGFP-N1/BMP2,pEGFP-N1/ IGF1-BMP2were constructed successfully with gene recombinant technique after identification with restriction enzyme.PartⅡ:The culture and the proliferation and differention of bone mesenchy-mal stem cellsThe bone mesenchymal stem cells(BMSC)were obtained aspirate from femoral bone and humeral bone of rat,then BMSc were cultivated in vitro.We observed thegrowth characteristics of BMSc under the microscope.We tested the survival and increase of BMSc growth by the relative data from MTT Method and detected the rate of ALP(+)by the improved Gomori's method.It the results that The adherent BMSc gave rise to colonies and grew quickly.The appearances of BMSc were not altered after several passages.Some colonies and cells locating in the center of colonles expressed significant ALP(+).The BMSc were obtained easily and The BMSc has high capability to proliferate and differentiate in vitro.PartⅢ:Expression and differentiation of BMP-2 and IGF-1 of cells cult- ure after bone mesenchyal stem cells transfected BMP-2 and IGF-1 in VitroDouble gene coexpression plasmid were transferred into bone mesenchyal stem cells by liposome in Vitro,fluorescence microscope observe cells culture after BMSCs transfected BMP-2 and IGF-1.fluorescence microscope observe the transfed cells.And detected the rate of ALP(+)by the improved Gomori's method and calcify nodus.It is the result that BMSCs,the transfed cells express masculine,pEGFP-N1/IGF1-BMP2 has the best transfection efficiency,and expressed signlficant ALP(+),calcify nodus formation.It indicated that cells culture after BMSCs transfected BMP-2 and IGF-1 can differentiate in vitro.Our findings indicate that:BMP2 can induce ectopic ossification and is an important factor of inducing ectopic ossification.IGF-1 can enhance the bone formation,and promote bone healing.IGF-1 increase ossification and elevate bone healing in coordination with BMP-2 The constructed pEGFP-N1/IGF1-BMP2 can besuccessfully transfected into BMSCs with cation liposome-med-iated transfection method.The exogenous BMP-2 and IGF-1 genes can be expressed constitutively in the transfected BMSCs.The lineage-committed different-tiation abilities of BMSCs containing combination of genes BMP-2 and IGF-1 is enhanced.The gene transfection technique in vitro can provide an experimental base for the modified seed cells in bone tissue engineering.