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目的 :比较 5 -氮胞苷 ( 5 -azacytidine ,5 -aza)于不同浓度、不同孵育时间对骨髓间充质干细胞 (mesenchymalstemcells,MSC)向心肌样细胞分化的影响 ;观察诱导后不同时间点心肌标志物的表达情况。方法 :体外分离培养Wistar大鼠MSC ,传代纯化后进行诱导实验。浓度分组 (C1-C4 ) :分别以 3、5、10、2 0 μmol/L5 -aza处理 2 4h ,之后更换新鲜培养基继续培养。时间分组 (T1-T5 ) :以 10 μmol/L分别孵育 6h、12h、2 4h、4 8h、72h ,之后更换新鲜培养基继续培养。各组均于首次加药 4周后行免疫细胞化学染色检测。RT -PCR检测 10 μmol/L5 -aza处理后 2、4、8周细胞GATA4mRNA、cTnTmRNA的表达情况。结果 :经 5 -aza诱导处理后 4周 ,C1-C4组Desmin及α -SarcomericActin染色阳性率各组比较均无统计学差异 (P >0 .0 5 ) ;而T1-T5各组其阳性率比较存在统计学差异 (P <0 .0 1)。RT -PCR显示 ,诱导后 2周 ,GATA4mRNA开始表达 ,持续至 8周 ;cTnTmRNA在诱导后 2周无表达、4周时弱表达 ,8周时更为显著。结论 :成年人鼠MSC在适当浓度 5 -aza处理后可以向心肌样细胞分化 ,在 3μmol/L～ 2 0 μmol/L范围内 ,5 -aza浓度的变化对MSC的分化率影响较小 ;在 4 8h内 ,适当延长 5 -aza的孵育时间可能增加MSC向心肌样细胞的转?
OBJECTIVE: To compare the effects of 5-azacytidine (5 -aza) on cardiomyocyte-like differentiation of mesenchymal stem cells (MSC) at different concentrations and different incubation times. The effects of 5-azacytidine Marker expression. Methods: MSC of Wistar rats were isolated and cultured in vitro and induced by passage. Concentration group (C1-C4): treated with 3,5,10,2 0 μmol / L 5-aza for 24 hours, then replaced with fresh medium and cultured. Time grouping (T1-T5): Incubation with 10 μmol / L for 6h, 12h, 24h, 48h, 72h, respectively, then replaced with fresh medium and continued to culture. All groups were detected by immunocytochemistry 4 weeks after the first dosing. The expression of GATA4mRNA and cTnTmRNA were detected by RT-PCR at 2, 4 and 8 weeks after treatment with 10 μmol / L 5 -aza. Results: The positive rate of Desmin and α -SarcomericActin in C1-C4 group showed no significant difference (P> 0.05) at 4 weeks after 5-aza treatment, while the positive rate of T1-T5 in each group There was statistical difference (P <0.01). RT-PCR showed that GATA4 mRNA began to express at 2 weeks after induction, and continued to 8 weeks. CTnT mRNA was not expressed at 2 weeks after induction and weakly at 4 weeks, and more significantly at 8 weeks. Conclusion: Adult rat MSCs can differentiate into cardiomyocytes after treatment with appropriate concentration of 5-aza. The changes of 5-aza concentration have little effect on the differentiation of MSCs in the range of 3μmol / L ~ 20μmol / L. 48h, the appropriate extension of 5-aaza incubation time may increase the MSC to the cardiomyocyte-like turn?