Effects Of Dimethyl Sulfoxide On The Differentiating Of P19 Cells To Cardiac Myocytes And Changes Of NADPH Oxidase Expression After The Differentiating

Objective ①To investigate the effects of different levels of DMSO on the induced differentiating of P19 cells to cardiac myocytes in vitro; ②Study the changes of NADPH oxidase after P19 cells differentiating to cardiac myocytes.Methods ①P19 cells were cultured with medium(containing 90% α-MEM, 10% FBS, penicillin 100u/ml, streptomycin 100 ug /ml), choose appropriate cell lines and digested with pancreatic enzyme, culture them in a 10 cm suspension culture dish with density of 1 x 106/ml, add 10 ml different levels induced culture solution(containing 0.4%, 0.9% and 1.4% DMSO) and suspension cultured. Exchange 7 ml culture solution at the 3th day. Absorb right amount of the embryoid body into tissue culture dishes when the cell aggregates formed at the 4th day, full exchange with culture solution after every 2 days. Culture them to the 13 th day and observe the cell morphology under a microscope at different growth period. Collect the induced cells at different times, Western Blot of Cardiac Troponin I(c Tn I) was used to identify cell differentiation and compare the differentiation of P19 cell culture with different levels of DMSO; ②Detect the level of subunit gp91-phox and p22-phox of NADPH oxidase before and after the differentiation with Western Blot; ③SPSS 11.0 statistical software was used to analysis the data, the levels of c Tn I, p22- phox and gp91- phox before and after the differentiation analysed with T test, and the results of Western blot analysed with software Image J, the level of test is P = 0.05.Results ①P19 cells which cultured by 0.4% DMSO were detected c Tn I-positive at thethe 9th day, the expression of c Tn I at the 11 th day was significantly higher than that at the 9th day, the 13 th day was higher than the 11 th day; cultured by 0.9% DMSO were detected c Tn I-positive at the 7th day, the expression of c Tn I at the 9th day was significantly higher than that at the 7th day, the 11 th day was higher than the 9th day, the 13 th day was higher than the 11 th day, P<0.05, all with statistical significance; apoptosis at 3th day which cultured with 1.4% DMSO, the induction was failed.②The expression of c Tn I which cultured with 0.9% DMSO was higher then P19 cells cultured with 0.4% DMSO at the same day with statistical significance(P<0.05). ③ The expression of gp91-phox and p22-phox in differentiated P19 cells were higher than in undifferentiated P19 cells, with statistical significance(P<0.05).Conclusion P19 cells can be induced to cardiac myocytes in vitro by DMSO with a certain condition; The differentiation efficiency of P19 cells to cardiac myocytes is related with the concentration of DMSO within limits and a high concentration may lead to apoptosis. The expression of gp91-phox and p22-phox were increased before and after the differentiation of P19 cells to cardiac myocytes, the level of NADPH oxidase and oxidative stress was increased.