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We established a cell culture model of osteoblast differentiation using MC3T3-E1 subclone 14 pre-osteoblast cells.Following the addition of different concentrations of a2,3-neuraminidase, cell surface a2,3-sialic acid was detected with fluorescein isothiocyanate (FITC)-labeled Maackia amurensis lectin (MAL-Ⅱ) and confirmed by flow cytometry.We observed positive alkaline phosphatase (ALP) staining cells, which indicated the formation of osteoblasts in MC3T3-E1 subclone 14 cells in 10 days culture.There were no significant changes following the addition of different concentration of a2,3 neuraminidase, with the exception that mineralized nodules were obviously decreased on day 14 by von Kossa staining.The characteristic biological markers and osteoblast-like cell-related factors of osteoblastic cells were examined at the mRNA level with reverse-transcriptase polymerase chain reaction (RT-PCR).Osteocalcin (OC) and osteopontin (OPN) transcript levels in the treated cells were not significantly different compared with the control group.However, mRNA levels of bone sialoprotein (BSP)、 osteoprotegerin (OPG) and vitamin D receptor (VDR) were obviously decreased.These results were supported by western blot analyses.The effect of 600mU/ml a2,3 neuraminidase on osteogenesis was similar to that of 200mU/ml a2,3 neuraminidase.