Objective To investigate the effect of the Notch-RBP-J signaling pathway on the phenotypic differentiation of RAW264.7 murine macrophage-like cells cocultured with B16F10 murine melanoma cells.Methods A small interference RNA (siRNA) targeting the CBF1/RBP-Jκ gene was designed.RAW264.7 murine macrophage-like cells were divided into four groups:unsilenced co-culture group cocultured with B16F10 cells,silenced co-culture group transfected with the designed siRNA and cocultured with B16F10 cells,blank control group cultured alone,positive control group induced by interleukin-4 (IL-4).After additional culture for different durations,Western blot,enzyme-linked immunosorbent assay (ELISA) and flow cytometry were conducted to determine the phenotype of macrophages,and reverse transcription (RT)-PCR was performed to detect the expressions of notch1,notch2,DLL1,DLL4 and Hes1 genes in macrophages.Statistical analysis was carried out by one-way analysis of variance (ANOVA),repeated measures ANOVA,linear trend test and the Bonferroni method with the SPSS17.0 software.Results Western blot showed that the relative expression level of CD163 in RAW264.7 cells was 1.016 ± 0.018 in the blank control group,1.274 ± 0.034,2.065 ± 0.094 and 3.615 ± 0.144 in the unsilenced co-culture group at 24,48 and 72 hours respectively,and 3.099 ± 0.071 in the positive control group,with significant differences between these groups (n =4,F =527.42,P ＜ 0.01).There was a significant increase in CD163 expression in RAW264.7 cells in the unsilenced co-culture group compared with the blank control group.As ELISA revealed,the levels of IL-10 in the culture supernatant of RAW264.7 cells were (167.61 ± 3.527),(433.433 ± 5.558) and (679.673 ± 8.101) ng/L in the unsilenced co-culture group at 24,48,and 72 hours respectively,significantly higher than those in the silenced co-culture group ((63.403 ± 0.856),(103.427 ± 2.072),(202.297 ± 3.61) ng/L,respectively,F =8.01,P ＜ 0.05).Western blot and flow cytometry both demonstrated a statistical reduction in the expressions of CD163 and CD206 in RAW264.7 cells in the silenced co-culture group compared with the unsilenced co-culture group (both P ＜ 0.05).The mRNA expressions of notch signaling pathwayrelated genes in RAW264.7 cells were also attenuated in the silenced co-culture group in comparison with the unsilenced co-culture group.Conclusions Although most of the RAW264.7 cells in the silenced co-culture group exhibited the M2 phenotype,their polarization toward M2 phenotype was weakened compared with those in the unsilenced co-culture group,implying that the activation of the Notch-RBP-J signaling pathway promotes the M2-polarization of RAW264.7 cells.