Differentiation Of Rabbit Bone Marrow Mesenchymal Stem Cells Into Osteoplasts With The Transfection Of The Chitosan Bearing PIRES-hVEGFl2lcDNA/hBMP-4

Objective To further study the isolation and cultivation procedures of mesenchymal stem cells from rabbit in vitro,and explore their biological properties;To build an experimental basis on applying MSCs as seed cells to treat many diseases such as bone defect;To identificate the correctness of pIRES-hVEGFl2lcDNA/hBMP-4; To investigate the feasibility of rabbit bone marrow mesenchymal stem cells differen- tiate into osteoplast with the transfection of the chitosan bearing pIRES-hVEGFl2l cDNA/hBMP-4 .Methods Bone marrow was drawn from rabbit femurs and bone marrow mesenchym- al stem cells were separated from bone marrow with percoll cell separation medium, the configuration was observed under phase—contrast microscope consecutively and the cell growth was measured by MTT method.Cell cycle and surface antigens were detected through flow cytometer.Recombinant coexpression plasmids of pIRES- hVEGFl2lcDNA/hBMP-4 were confirmed by restriction enzymolysis and transferred rabbit MSCs by chitosan,then bone marrow mesenchymal stem cells were induced to differentiate into osteoplast with the transfection of the chitosan bearing pIRES- hVEGFl2lcDNA/hBMP-4.The differentiation result was detected by alkaline phos- phatase staining and alizarin red vital staining,And the functional change of the cells was Observed under scanning electronic microscope.Results Lots of bone marrow mesenchymal stem cells were separated from bone marrow with Percoll cell separation medium, the subcultured cells showed active proliferative ability.More than 91% of subceltured MSCs were adhesive in 12h. MSCs were positive for CD29,CD90 and negative forCD34.After 14 days of trans- fection with the chitosan bearing pIRES-hVEGFl2lcDNA/hBMP-4, the alkaline phosphatase staining and alizarin red vital staining of the experiment groups were positive while the contrast groups were negative. There were plenty of alkaline phos- phatase vesicle in the cells and great amount of stroma secreted around the cells in the experiment groups,while there were almost nothing secreted in the contrast groups.Conclusion Rabbit MSCs showed stable and rapid growth by isolating and cultivating in vitro in eligible culture condition.The characteristics make it possible to MSCs to be the seed cells for tissue engineering.Bone marrow mesenchymal stem cells can differentiate into osteoplast with the transfection of the chitosan bearing pIRES- hVEGFl2lcDNA/hBMP-4.