Construction of BMP_(4/7) fusion gene adeno-associated virus and biological effects of transfection on bone marrow stromal cells in rabbits / 中国矫形外科杂志

ABSTRACT

[Objective]To determine the biological effects of transfection of adeno-associated rirus(AAV) BMP4/7 on rabbit bone marrow stromal cells (BMSCs).[Method]The mature peptide of BMP4 and BMP7 were gained by One-Step RT-PCR from the human placenta and the BMP4/7 fusion gene was gained through gene recombinant techniques and then transferred to pGEM plasmid. The BMP4/7 fusion genen was cut down from the pGEM-BMP4/7 and the recombination was successfully completed in colibacillus, and recombinant adeno-assosiated was produced in 293 cells. Rabbit BMSCs were transfected with the recombinant adeno-assosiated virus vectors carrying BMP4/7 fusion gene with differen multiplicitv of infection(MOI) values. Cell growth curves were drawn to evaluate the biologic effect of AAV-BMP4/7 on cyto-activity. The transfection efficiency was measured using MTT method. The ossification of cells was evaluated by investigating the shape change of the cell ability of ALP and OC at 7,14 days after transfection. Cells were then transfected with AAV-BMP4/7 and AAV-EGFP,respectively.[Result]1.We successfully constructed the recombinant adeno-assosiated virus with BMP4/7 fusion gene.2.The ttransfection efficiency of AAV-BMP4/7 was roughly 72% without siginifficant biologic effect on cyto-activity.The ossification of cells was significant after transfection with AAV-BMP4/7. The 1?105 vg/cell MOI value of transfection efficiency was stroger than 5~104 vg/cell MOI value (59.3,8%). 3. The concentrations of ALP and OC in AAV-BMP4/7 transfection groups were significantbly higher than in AAV-EGFP groups (tALP=896.88 P