Comparision of Overexpression of Cbfa1 and BMP2 in Fibroblast NIH3T3

Objective: to study and compare the function of overexpression of core binding factor á1 (Cbfa1) and bone morphogenetic protein2 (BMP2) on osteoblast related genes in fibroblast NIH3T3, so as to find which is more useful to induce NIH3T3 to differentiate into osteoblast-like cell. Methods: Cbfa1 and BMP2 recombinant retroviral plasmid vector--pLEGFP-IRES-Cbfa1, pLEGFP-IRES-BMP2 were constructed. Then the retroviral plasmids were transfected into the packaging cells PT67 by lipofectin method and selected by G418 to obtain stable virus-producing cell lines. The medium from packaging cells containing virus were added to fibroblast NIH3T3, G418 was used to choose the stable overexpressing Cbfa1 or BMP2 cells. Real-time PCR, western blot and mineralization assay were adopted to determine the expression of Cbfa1, BMP2, osteoblast related genes and the biomineralization function of the infected NIH3T3. Results: Real-time PCR and western blot confirmed that the infected NIH3T3 were expressing Cbfa1 or BMP2 gene. Both in Cbfa1 and in BMP2 overexpressing NIH3T3, the expression of osteoblast related genes (OPN, BSP, OC, type I collagen) could be detected and In vitro nodule size and quantity were more than the control cells. While the expression of osteoblast related genes and nodule formation in Cbfa1 overexpressing cells seems more significant than that in BMP2 overexpressing cells. Conclusion: Overexpression of Cbfa1 or BMP2 in NIH3T3 cells both can induce NIH3T3 to differentiate into osteoblast type by inducing osteoblast related genes expression, while Cbfa1 seems more function than BMP2 by this method in NIH3T3 in vitro.Supported by National Natural Scientific Foundation of China.30371537