Retrovirus-mediated expression of Indian hedgehog in c3h10t1/2 cells and osteogenic potential

BACKGROUND: Indian hedgehog (Ihh) has been confirmed to play an important role in regulating bone growth by a great amount of studies. OBJECTIVE: To clone the Ihh recombinant retrovirus vector, in addition, to observe its expression in C3H10T1/2 cells and study the effect on the osteogenesis METHODS: According to the molecular biological technology, the Ihh gene sequence was linked into the retroviral vector pSFG vector which also contained the enhanced green fluorescence protein (EGFP) gene sequence. Viral supernatants harvested from package cell line were transfected into C3H10T1/2 cells. Then, the intensity of EGFP and the efficiency of gene transfer were monitored under the confocal microscope. The level of Ihh expression was detected by Western blot and morphologic changes of C3H10T1/2 cells were observed. Alkaline phosphatase staining was also used to determine the osteogenic differentiation. RESULTS AND CONCLUTION: The results of the confocal microscope and Western blot show that Ihh and EGFP proteins are coexpressed in a high level in C3H10T1/2 cells. The Ihh affects the morphology of C3H10T1/2 cell line significantly and induces the alkaline phosphatase expression in vitro. Therefore, C3H10T1/2 cells have the potential capability of differentiating into osteocytes.