Objective To establish a co-culture system to observe the interactions between mouse macrophage RAW264.7 and myoblast C2 C12 cells. Methods RAW264.7 and C2 C12 cell lines were co-cultured in a Transwell chamber containing conditioned medium for myogenic differentiation

and the changes in the cell morphology were observed under a phase-contrast microscope. On days 1

3 and 5 of co-culture

the cell proliferation was assessed with CCK-8 assay

and the protein expressions of Myf5

MyoD

myogenin

iNOS and Arg-1 were detected using Western blotting; the concentrations of interleukin-1β(IL-1β) and IL-10 in the supernatant were detected using enzyme-linked immunosorbent assay(ELISA). Results Co-culture with RAW264.7 cells obviously accelerated the myogenic differentiation and promoted the formation of multinucleated myotubes in C2 C12 cells. Compared with the cells cultured alone

C2 C12 cells in the co-culture system showed significantly inhibited protein expression of Myf5(P<0.05) and enhanced expressions of MyoD(P<0.05) and myogenin(P<0.01) on day 1

and inhibited expression of Myf5(P<0.01) and increased expression of MyoD(P<0.01) on day 3; significantly lowered cell viability(P<0.05) and Myf5 expression(P<0.01) with increased area of myotubes(P<0.01) were observed on day 5. The co-culture did not produce significant effect on the morphology of RAW264.7 cells

but caused significant inhibition of iNOS expression(P<0.01) and IL-1β secretion(P<0.05) and increased Arg-1 expression(P<0.01) on day 1 of the co-culture; significantly lowered iNOS expression and IL-1β secretion(P<0.01) and increased Arg-1 expression(P<0.01) and IL-1β secretion(P<0.01) occurred in RAW264.7 cells on day 3 of the co-culture

and on day 5

the cells showed more obvious clustering with significantly enhanced proliferation(P<0.05)

lowered iNOS expression and IL-1β secretion(P<0.01)

and increased secretion of IL-10(P<0.05). Conclusion Co-culturing C2 C12 cells with RAW264.7 cells accelerates the myogenic differentiation of the former and promotes the proliferation and M2 polarization of the latter.