Objective To investigate the effects of vascular endothelial growth factor(VEGF)on the phenotypic polarization level and osteoclast differentiation of RAW264.7 macrophages under lipopolysaccharide(LPS)induction.Methods RAW264.7 cells in logarithmic growth phase were divided into 3 groups,with a culture period of 4 days.Control group:RAW264.7 cells were in normal culture.LPS group:100 ng/mL LPS was added at the same time as each fluid change after cell inoculation and adherence.LPS+VEGF group:LPS was added as the same as LPS group,1 μg/mL VEGF was added at the fluid change on day 4.RAW264.7 cells were cultured in gradient density with toluidine blue staining to clarify the appropriate planting density.In each group,the morphological changes of cells were observed under a microscope,the expression levels of interleukin(IL)-12,IL-10 and arginine-1(Arg-1)protein were detected by Western blot,the polarization levels of macrophages were determined by flow cytometry,and the differentiation levels of osteoclasts were detected by tartrate-resistant acid phosphatase staining.Results Toluidine blue staining clarified that the cell planting density was 2.5×103/cm2.Microscopic observation showed a large number of M1-type macrophages in LPS group and a small number of M2-type macrophages in LPS+VEGF group.IL-12 protein expression in LPS+VEGF group was lower than that in LPS group(P＜0.05),while IL-10 and Arg-1 protein expression were significantly higher than those in LPS group(P＜0.05).The positive expression rate of F4/80+CD206 protein in LPS+VEGF group was higher than that in LPS group(P＜0.01),and the positive staining count of osteoclasts was higher than that in LPS group[(41.83±3.25)vs(25.67±4.89),t=6.154,P＜0.01].Conclusion VEGF can promote LPS-induced polarization of RAW264.7 cells to the M2 phenotype and promote osteoclast differentiation.
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