5-Azacytidine combined with angiotensin Ⅱ induces differentiation of adipose-derived mesenchymal stem cells into cardiomyocytes

Wang Jing; Han Jiang-hong; Li Qiong

doi:10.3969/j.issn.2095-4344.2017.29.007

Abstract：BACKGROUND:Taking effective measures for myocardial necrosis develops a trend in the clinical treatment of myocardial infarction.OBJECTIVE:To investigate the mechanism of 5-azacytidine combined with angiotensin Ⅱ in the differentiation of mouse adipose-derived mesenchymal stem cells into cardiomyocytes.METHODS:Twenty ICR mice aged 4 weeks old were used to isolate adipose-derived mesenchymal stem cells using type Ⅰ collagenase digestion method.Cell morphology was observed,cell proliferation was detected using MTT,and immunofluorescence technique was used to detect the expression of CD90,CD105,CD73 and CD45.Passage 4 adipose-derived mesenchymal stem cells were cultured for 24 hours,then induced by 15 μmol/L 5-azacytidine or 15 μmol/L 5-azacytidine plus 0.1 μmol/L angiotensin Ⅱ for another 24 hours,and finally cultured in a complete medium.The expression of cTnT protein in myocardial cells was detected by western blot assay.RESULTS AND CONCLUSION:Primary adipose-derived mesenchymal stem cells with small nuclei were spindle-shaped or starlike.After passage,the cells were increased in size,with uniform shape and regular arrangement.Passage 3 mouse adipose-derived mesenchymal stem cells showed an S-shaped growth curve:days 1-3 was incubation period,the cells showed logarithmic growth at day 4,the cell growth reached the peak at days 5-7,and then entered stationary phase.The passage 4 cells were positive for CD90,CD105 and CD73,but negative for CD45.The expression of cTnT was significantly higher after cell induction with 5-azacytidine combined with angiotensin Ⅱ than 5-azacytidine alone.In conclusion,the combined use of 5-azacytidine and angiotensin Ⅱ is beneficial for the cardiomyocyte-like differentiation of adipose-derived mesenchymal stem cells.