The Study Of Vascular Endothelial Grouwth Factor Express In Embryonic Osteogenic Cells

Objective:Investigate cultruring embryonic osteogenic cells contains vascular endothelial growth factor(VEGF)gene in vitro,and then amplificate it back into replantation bady. Observing the transfected osteoblast's proliferation and regeneration.then taking the transfected osteoblast cells which contain purpose gene as seeds cell.On the basis of selected absorbing material,through biofilm formation scheduled three-dimensional shape.Planting cartilage cell which are cultured in vitro on its surface,and make them compatible. covering biological membrane(degradation materials)on the surface,to construct recombination tissue engineering bones. Expect to treat osteonecrosis at the molecular level providing,a new rationale and technique for clinical treatment of bone necrosis.Now clinical work in various causes of bone necrosis increase in the number of cases,and there is more varieties,the trence gradually becomes younger.At present the clinical treatment of bone necrosis commonly uses methods:filling Sigu prosthesis removal,replacement prosthesis,pedicled bone grafts,Chinese medicine conservative treatment,etc,these therapies are often greaterly injuried but the results is not ideal,the patient's movement and sensory function is often not quite satisfied.Even cause lifelong disability.Thus the purpose of the designation to this subject is through modem molecular biology technique,take the VEGF -gene fragment as purpose gene,integrate with type5 adenovirus vector,constructing adenovirus vector with purpose gene.Amplificating embryonic osteogenic cell(SD rat)in vitro.When cell multiplication enter to increased logarithmic phase,transfected the purpose cells.To make the embryonic osteogenic cell not only keeping the activity of reproduction,but also promoting blood vessel regeneration through the expressing of VEGF.As a result,the problems of innostosis and ischemia both are solved. Immunohistochemistry and immunofluorescence staining qualitative analyze the expression of transfected genes in target cells.And quantitative detect osteoblast cells in the gene transfection by flow cytometry.through cell multiplication experiment(describing the curve of cell growth before and after transfection)to detect the biologic activity and multiplication metergasis.After target cell gene modifing and well expressing, transplant back into the donator;observe the vascularize proceeding of transplant and multiplication of transfected embryonic osteogenic cell.through observing the internal environment of animal model which is bone coloboma and the biology condition of tissue engineering bone in various kinds of stress.expect to make sure that this way is a n effective feasible Ideal bionicstreatment to cure various kinds of clinical osteonecrosis.Providing a new and breakthrough for clinical osteonecrosis treatment.Methods:The materials of primary culture is fetal rat's cranium,the routine conditions is 37℃,saturation humidity 5% CO2,the skeletogenous will be cultured to entere the logarithmic growth phase cells after before experimental.1 Cell culture:take the new born SD rats in 75%alcohol soaked in 5 minutes.Limbs fixed in wax disc.Cutdown the skin and exposed the calvarial and removed the it,as fast as possible clean out the remnants of the periosteum and soft tissue,then immersed and rinsed in Hanks liquid.Using trypsin and typeⅠcollagenase of double enzymatic digestion digest for 30 min,immersed and rinsed in Hanks liquid,cut the organization into 1 mm~3 in plate,Adherent cultured in DMEM, observe the growth of the cell by re-inverted microscope everyday.2 Osteoblast function identification and detection technologies,including the observation of(osteoblasts scanning electron microscope),Giemsa staining,alkaline phosphatase (ALP),mineralized nodule staining(Alizarin red staining),and other methods Identification.3 Construction of typy 5 adeoviruse vetor containing VEGF gene fragment(ad5-h-VEGF),cultured and amplificated the embryonic osteoblast cells in vitro,passages to enter logarithmic growth phase.then transfected into the targe of osteoblast cells.Mapping their growth curve before and after transfection respectivly.Immunohistochemistry and immunofluorescence staining qualitative analyze the expression of transfected genes in target cells.And quantitative detect osteoblast cells in the gene transfection by flow cytometry.Results:1 culture and observation of osteoblast:primary osteoblasts crawling all the culture bottles in about 5-7 days, inverted microscope observing the growing adherent cells, most cells are fusiform shape or triangle.round or ellipticalcell nucleus state in the middle of the cells,or in one side of the cells. most of the nucleuss have several apophysis,one or more nucleolus.cytoplasm is abundance and clear.Cell division usually appears in growing period,cell process conjugated with each other.There are multiformity cell process to stretch out the cell surface.cells are growthing in the way of colony,and stratums,to form cell tubercles or cellbolus.2 Scanning electron microscopy:osteoblasts have many processes on the surface,and interconnected by the processes.3 Alkaline phosphatase(ALP)staining(Gomori calcium cobalt),and the cytoplasm in the osteoblasts showed black ash particles or bulk precipitation.4 Mineralized nodule staining(alizarin red),mineralized nodule and different red clumps are observed by naked eye.5 Giemsa staining,abundant cytoplasm dyed pink,nuclei stained purple blue,round or oval state in the central or side, nucleolus are showed obviously.6 VEGF transfection before and after control cell growth curve:after transfection osteoblast proliferated faster than non-transfected,at different times,have enhanced proliferation trends,and after a certain time growth became more gentle.7 Purpose gene of osteoblast after transfection immunohistochemical staining with hematoxylin-after qualitative analysis: a different infection multiple Ad5-h-VEGF infected osteoblast cells,cultured for 72 hours,the control group that the transfected cells(-)group,the red cell cytoplasm not color.Transfection is the experimental group(+)group,transfected cells were red nuclei,cytoplasm was red or brown soil.8 Qualitative analysis of immunofluorescence staining with PE-after,after transfected purpose gene,:Ad5-h-VEGF infected osteoblast Cells in different MOI,cultured for 72 hours,the control group that the transfected cells(-)group nucleus showed red fluorescence,cytoplasm showed no significant color.the experimental group that the transfected cells(+)group, transfected cell nucleus showed red fluores- cent,cytoplasm was pale green fluorescence.9 AdS-h-VEGF infected osteoblast cells in different MOI, cultured for 72 hours,flow cytometry detect the gene transfection positive rate.quantitative analyze of the transfected purpose gene.Conclusion:1 Through the double enzyme digestion,adherent culture can get more pure primary osteoblasts.Through the way of morphology and growth characteristics,sclerotomal cells can be identificated.and could be verified by alkaline phosphatase (ALP)staining(Gomori calcium cobalt),mineralized nodule staining(alizarin red),Giemsa staining method according to osteoblasts' biochemical characteristics.2 Adenovirus vector carrying the target gene-VEGF gene(ad5-h-VEGF),can successfully transfected osteoblasts cells,and the transfection efficiency to a certain extent,in a direct ratio trend of dose-time,the best transfection MOI(plural infection)is about 45,the best time is about 48-72 hours after transfection.3 Ad5-h-VEGF vector can successful transfected osteoblasts cells,and VEGF can successful express in osteoblast cells.and it is belongs to cytoplasmic expression.After transfected with VEGF,cells can promote cell growth and bone formation.4 VEGF in the expression of osteoblast cells can be qualitative detected by immunohistochemistry,immunofluorescence, quantitative detectedn by flow cytometer.