The Effect Of FPR1 And Fullerene Derivatives On The Differentiation Of Adipose Derived Stem Cells And Bone Marrow Macrophages

OBJECTIVETo investigate how formyl peptide receptor 1(FPR1)gene and a fullerene derivative F2 affect differentiation of mouse and human Adipose Derived Stem Cells and mouse Bone Marrow Macrophages.METHODS1.Isolated adipose derived stem cells and bone marrow macrophages from C57BL/6 wild type mouse and FPR1 knockout mouse.Cultured human adipose derived stem cells.2.Treatment for cells:Part ?:The Effect of FPR1 and Fullerene Derivative F2 on the Adipogenic and Osteogenic Differentiation of Adipose Derived Stem Cells.Blank control groups were set as DMEM culture medium supplemented with 10%fetal bovine serum(FBS)and 100?g/ml hybrid antibiotics(penicillin and streptomycin,P/S).Positive control groups were set as adipogenic medium(AM)or osteogenic medium(OM).Treatment groups were set by adding 0.2?g/ml fullerene derivative F2 on the basis of positive control groups.Part ?:The Effect of FPR1 and Fullerene Derivatives on the Differentiation of Bone Marrow Macrophages.Blank control groups were set as RPMI 1640 culture medium supplemented with 10%fetal bovine serum(FBS),100?/ml hybrid antibiotics(penicillin and streptomycin,P/S)and 20ng/ml macrophage colony-stimulating factor(MCSF).Positive control groups were set by adding 100ng/ml lipopolysaccharide(LPS)or 150ng/ml receptor activator for nuclear factor-Kappa B ligands(Rankl).Treatment groups were set by adding 0.2?g/ml fullerene derivative F2 on the basis of positive control groups,but F2 was added to the culture 0.5 h prior to addition of LPS or Rank1.3.Measurement and evaluationPart ?;At different time points of cell culture,cell mineralization or lipid droplet formation was evaluated by alizarin red staining(ARS)or oil red O staining.The expression level of adipogenesis or osteogenesis genes was determined by RT-PCR.Cell counts by WST-1 kit and crystal violet staining were used to evaluate the cell number,thus to evaluate the cytotoxicity of F2.Part ?:After stimulation,cell counts by WST-1 kit were used to evaluate the cell number,thus to evaluate the cytotoxicity of F2.The expression level of inflammatory or osteoclastogenesis genes was determined by RT-PCR.Nitrite production was assessed by commercial nitrite kit.RESULT1.The capability of adipogenic differentiation of FPR1 knockout mouse ADSCs was superior than that of C57BL/6 wild type mouse ADSCs,but the capability of osteogenic differentiation of FPR1 knockout mouse ADSCs was inferior than that of C57BL/6 wild type mouse ADSCs.2.Pro-inflammatory gene expression of FPR1 knockout mouse BMMs induced by LPS was more than that of C57BL/6 wild type mouse BMMs.Osteoclastogenesis gene expression of osteoclastogenic induced FPR1 knockout mouse BMMs was more than that of C57BL/6 wild type mouse BMMs.3.F2 could inhibit lipid droplet formation during adipogenic differentiation and cell mineralization during osteogenic differentiation.4.NO production and pro-inflammatory gene expression induced by LPS were significantly diminished by F2 in C57BL/6 wild type mouse BMMs.F2 could promote osteoclastogenic differentiation induced by Rankl and MCSF in C57BL/6 wild type mouse BMMs.CONCLUSION1.FPR1 can promote osteogenic differentiation and inhibit adipogenic differentiation of mouse ADSCs,embodied in lipid droplet formation inhibition and cell mineralization promotion.The expression of FPR1 gene is upregulated after osteogenic induction.2.F2 can inhibit adipogenic and osteogenic differentiation of mouse ADSCs,embodied in lipid droplet formation and cell mineralization inhibition.3.F2 can inhibit inflammation,embodied in reducing ROS production and down-regulating the expression of inflammatory genes.F2 can promote osteoclastogenic differentiation of mouse BMMs,embodied in up-regulating the expression of osteoclastogenesis genes.4.FPR1 can inhibit the inflammation process and osteoclastogenic differentiation of mouse BMMs,embodied in upregulation of the pro-inflammatory gene expression of FPR1-deleted BMMs after inflammtion induction and upregulation of the osteoclastogenesis gene expression after osteoclastogenic induction.