Differentiation of monolayer-cultured P19 cells toward cardiomyocytes induced by 5-azacytidine
AIM: To research the effect of Nkx2-5 gene on the myocardial differentiation of monolayer-cultured P19 cells induced by 5-azacytidine(5-aza). METHODS: The experiment contains two groups, experimental group and control group. The cells of experimental group were P19 cells which expressed Nkx2-5 gene constantly, while the cells of control group were normal P19 cells. Under the monolayer-culture condition, the cells of both two groups were induced by 5-aza with a concentration of 1 μmol/L. The growth state of cells were observed by inverted microscope. At 4th, 8th, 12th and 16th day, the expression of α-sarcomeric actin(α-SA) and cardiac troponin T(cTnT) was detected with immunocytochemical methods. The ultrastructural changes of cells were observed at 16th day after induced by 5-aza. RESULTS: In experimental group, α-SA and cTnT expression was detected at 8th, 12th and 16th day after induced by 5-aza and the expression of the two proteins was gradually increased. In control group, the expression of the two proteins was not detected. In experimental group, observation of the ultrastructure showed that cell junction appeared between adjacent cells and some of the adjacent cells extended mutual chimeric processes which were similar to the early myocardial intercalated disk-like structure. In the cytoplasm of some cells, some dense, myofilament-like structure arranged in parallel way was found. No differentiated cell was found in control group. CONCLUSION: Myocardial differentiation from monolayer-cultured P19 cells can be promoted by exogenous expression of Nkx2-5 gene.
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