5-氮杂胞苷联合血管紧张素Ⅱ诱导脂肪间充质干细胞向心肌细胞的分化

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

背景:采取有效措施修复坏死心肌成为临床治疗心肌梗死的新方向。目的:探讨5-氮杂胞苷联合血管紧张素Ⅱ对小鼠脂肪间充质干细胞向心肌细胞分化的影响。方法:取20只4周龄ICR小鼠,利用Ⅰ型胶原酶消化法获得脂肪间充质干细胞,观察细胞的形态学特点,运用MTT法检测细胞的増殖情况,运用免疫荧光技术检测细胞表面抗原CD90,CD105,CD73,CD45的表达。第4代脂肪间充质干细胞培养24 h后,更换为含15μmol/L 5-氮杂胞苷和15μmol/L 5-氮杂胞苷+0.1μmol/L血管紧张素Ⅱ的培养基诱导24 h,然后更换为完全培养基,Western Blot检测细胞中c Tn T蛋白表达。结果与结论:①原代脂肪间充质干细胞胞核较小,呈梭形、星形,传代后细胞体积增大,形态趋于一致,排列规则;②第3代小鼠脂肪间充质干细胞的生长曲线呈现"S"形,第1-3天为潜伏期,第4天进入对数生长期,第5-7天细胞生长达到峰值,进入平稳期;③第4代脂肪间充质干细胞表面CD90、CD105及CD73呈阳性表达,CD45阴性表达;④5-氮杂胞苷联合血管紧张素Ⅱ组心肌细胞中c Tn T蛋白表达量显著高于5-氮杂胞苷(P<0.05);⑤结果表明,5-氮杂胞苷联合血管紧张素Ⅱ能够显著促进脂肪间充质干细胞向心肌细胞样细胞分化。

BACKGROUND:Taking effective measures for myocardial necrosis develops a trend in the clinical treatment ofmyocardial infarction.OBJECTIVE:To investigate the mechanism of5-azacytidine combined with angiotensin II in the differentiation of mouseadipose-derived mesenchymal stem cells into cardiomyocytes.METHODS:Twenty ICR mice aged4weeks old were used to isolate adipose-derived mesenchymal stem cells usingtype I collagenase digestion method.Cell morphology was observed,cell proliferation was detected using MTT,andimmunofluorescence technique was used to detect the expression of CD90,CD105,CD73and CD45.Passage4adipose-derived mesenchymal stem cells were cultured for24hours,then induced by15μmol/L5-azacytidine or15μmol/L5-azacytidine plus0.1μmol/L angiotensin II for another24hours,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 werespindle-shaped or starlike.After passage,the cells were increased in size,with uniform shape and regular arrangement.Passage3mouse adipose-derived mesenchymal stem cells showed an S-shaped growth curve:days1-3wasincubation period,the cells showed logarithmic growth at day4,the cell growth reached the peak at days5-7,and thenentered stationary phase.The passage4cells were positive for CD90,CD105and CD73,but negative for CD45.Theexpression of cTnT was significantly higher after cell induction with5-azacytidine combined with angiotensin II than5-azacytidine alone.In conclusion,the combined use of5-azacytidine and angiotensin II is beneficial for thecardiomyocyte-like differentiation of adipose-derived mesenchymal stem cells.