Objective   To explore the possibility and efficiency of the myocardial differentiation of P19 cells that exogenous expressing Nkx2-5 gene under monolayer culture condition with 5-azacytidine (5-aza) induction.    Methods   P19 cell line was divided into 2 groups

experimental group and control group. The cells of experimental group were P19 cells which expressed Nkx2-5 gene constantly. While control group were common P19 cells. Under the monolayer culture condition

the cells of 2 groups were induced by 5-aza at a concentration of 1 μmol/L. The growth state of cells were observed by inverted microscopy. In 4

8

12 and 16 d after induction

Western blot analysis was used to detect the expression of α-sarcomeric actin and cTnT. The co-expression of α-sarcomeric actin and cTnT was detected with immunofluorescence methods.    Results   With 5-aza inducing monolayer culture

the cells of experimental group and control group all grew slowly. The number of dead cells was increased. As the attached cells increased in number

they gathered and formed a state similar to adherent aggregates-like structure. The cells in the center of the aggregates were small and dense

while the surrounding cells became longer

changed into spindle-like shape with extended processes and arranged in radial way. In experimental group

the expression and co-expression of α-sarcomeric actin and cTnT were detected in 8

12 and 16 d after 5-aza induction. The expressions of the 2 proteins were gradually increased. Statistical analysis showed that the expression of α-sarcomeric actin in 16 d and 12 d was significantly increased compared with that in 8 d (P0.01). The expression of α-sarcomeric actin in 16 d was significantly higher than that in 12 d (P0.01). The expression of cTnT in 16 d and 12 d was significantly higher than that in 8 d (P0.01). In control group

the expression of the 2 proteins was not detected.    Conclusion   5-aza induces monolayer cultured P19 cells with exogenous expression of Nkx2-5 gene to cardiac differentiation.