Experimental Study On Bone Differentiation Of Rabbit Bone Mesenchymal Stem Cells By Gene Transfection Of Insulin Like Growth Factor-1

AIM: Used the density gradient centrifugation combined with attachment culture method to isolate and obtain the bone marrow mesenchymal stem cells (BMSCs) of rabbits in vitro,and by using the Lipofectamine2000 to transfect the pcDNA3.1-IGF-1 into the BMSCs to observe the bone formation effects of Insulin like Growth Factor-1(IGF-1) gene,in order to lay a foundation for the further study of bone formation by transfer of IGF-1 gene into BMSCs.Methods: 1.(1) Using gradient centrifugation combined with attachment culture method to isolate and obtain the bone marrow mesenchymal stem cells of rabbits in vitro;(2) osteoblast-differentiaton of rabbit bone marrow mesenchymal stem cells in vitro and observed by invert microscope.2.(1) Amplification and purification of the pcDNA3.1-IGF-1 DNA plasmid;(2) transfecting the IGF-1 gene into BMSCs by using the Lipofectamine2000,and identificated the expression of IGF-1 by using the immunofluorescence,RT-PCR,and Western blot;(3) drawed and analysized the BMSCs growthing curve which transfected with IGF-1 by using the MTT colorimetric assay;(4) used the ELISA,ALP and Von Kossa to observe the bone formation effects in vitro of BMSCs which transfected by IGF-1 gene.Results:1.(1)Success of the isolation culture of rabbit bone marrow mesenchymal stem cells;(2) the induced rabbit bone marrow mesenchymal stem cells have expressed a higher level of the ALP and the node of calcium;2.(1) amplification and purification of plasmid DNA of pcDNA3.1-IGF-1 were success;(2) stable transfection of IGF-1 gene into BMSCs was obtained;(3) MTT showed the BMSCs has a higher reproductive activity after gene transfection;(4) the pcDNA3.1-IGF-1 transfected BMSCs could show a higher potentiality of bone formation through the ALP observing and calcium node counting (p<0.05). Conclusion:1.The combination of gradient centrifugation and attachment culture method was an effective method to isolate BMSCs which had a higher proliferation ability;2.the pcDNA3.1-IGF-1 transfected BMSCs showed a better potentiality of bone formation in vitro,and colud lay a foundation for the further study of bone formation by IGF-1 gene therapy.