Objective:To study the role of Nkx2-5 gene in 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells.Methods:The experiment was divided into two groups:an experimental group and a control group.The cells in the experimental group were P19 cells stably expressing Nkx2-5 gene, and cells in the control group were P19 cells.Under the monolayer-culture condition, the cells of two groups were induced by 5-azacytidine (1 μmol/L).The growth of cells were observed by inverted microscope.On the 4th day, 8th day, 12th day and 16th day after induction, RT-PCR was used to detect the expression of GATA-4, α-MHC and ANP gene.Results:In control group, there was no ANP expression after induction; GATA-4 expression was seen on the 8th day, 12th day, and 16th day after induction; and α-MHC expression was found on the 12th day and 16th day.In experimental group, the expression of GATA-4 was detected on the 4th day, 8th day, 12th day and 16th day after induction; Alpha-MHC and ANP expression was noticed on the 8th day, 12th day and 16th day after induction.RT-PCR results showed that the expression of GATA-4 and α-MHC in the experimental group was earlier than that in the control group.And at all time points of observation, the expression of GATA-4 and α-MHC in the experimental group was significantly increased compared with that in the control group(P<0.05,P<0.01), except for α-MHC expression on the 12th day.Conclusion:Nkx2-5 gene can promote 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells.