Studies On Repair Of Bone Defect By Mouse Sarcoblast Transfected By Adenovirus-mediated Core Bindind Factor α-1

Objective To clone human osteogenic sarcoma core binding factorα1 gene cDNA full length and generate high titers level recombinant adenoviruses containing hCbfal gene the replication defective recombinant adenoviruses AdEasy system was used. To induce expression of osteogenic phenotype by mCbfa1 gene the mouse C2C12cells were infected by recombinant adenovirus. Then the tissue-engineered bone was constructed by combination of infected C2C12 andchronOS and its feasibility and ability to enhance segmental bone defect healing of mouse femur was explored.Methods (1) mCbfal gene was cloned into Shuttle vector to generate pAdTrack-CMV-Cbfal, which was linearized by PmeI and co-transformed into adenoviral backbone plasmid competent cells-AdEasier-1 Cells to obtain recombinant adenoviral plasmid- pAd-Cbfa1 by homologous recombination. Recombinants were selected and confirmed by restriction endonuclease analysis. After packed in 293 cells, the recombinant adenoviruses AdCbfal were generated. (2) The proliferation and cell cyle analysis of gene-modified C2C12 were assessed by MTT method and FCM after transfected with Ad-Cbfa1. The methods of RT-PCR, immunohistochemical staining and western blot were used to test the expression of Cbfa1 protein. Gomori stain and ALP testing kit were used to measure ALP activity and immunohistochemistry testing kit were used to measure OCN contents. In addition, the collagen type I were tested with the methods of RT-PCR and immunohistochemical staining. (3)...