骨髓基质干细胞心肌化诱导过程中转录因子NKx2.5的表达

Time Course of NKx2.5 Expression in Bone Marrow Stromal Cells to Myocardial Differentiation Induced by 5-aza /DMSO

目的 :研究 5 -氮杂胞嘧啶 (5 -azacytidine,5 -aza)和二甲亚砜 (dimethylsulfoxide ,DMSO)诱导人骨髓基质细胞 (humanbonemarrowstromalcells ,hBMSC)心肌分化过程中心肌特异转录因子NKx 2 .5表达的变化。方法 :取传至第 8代hMSC ,以3μmol/L的 5 -aza和DMSO诱导 2 4h。 1周后重复诱导 1次。在诱导后 1、2和 3周 3个时间点以半定量RT -PCR方法检测细胞心肌特异转录因子NKx 2 .5的表达变化。结果 : hMSC经 5 -aza诱导后 ,约 1周左右细胞明显变大 ,2周时细胞走向趋于一致 ,邻近细胞间连接较前紧密 ,并形成肌节样结构。RT -PCR的结果显示 ,5 -aza可诱导心肌特异转录因子NKx 2 .5 ,并一直持续表达 3周。在DMSO诱导hBMSC后 1周 ,开始可检测到细胞发生转录水平变化NKx 2 .5表达 ,并均可持续表达至诱导后 3周 ;NKx 2 .5表达量有随诱导后时间延长而增加的趋势。结论 :5 -aza和DMSO在体外可诱导心肌特异转录因子NKx 2 .5的表达 ,从而启动hMSC心肌化的过程。

Objective: To investigate the time course of NKx2.5 expression in bone marrow stromal cells to myocardial differentiation induced by 5-aza /DMSO. Methods: Human bone marrow was aspirated from iliac of healthy adults. hBMSC were isolated using Percoll gradient centrifugation method. Cells were cultured in L-DMEM with FGF (0.5μg/L) at 37℃ in humid air with 5% CO 2 . The specific surface antigens of the mesenchymal stem cell were assayed by FACS after the 2nd to 8th passage. Cardiomyocyte differentiation was induced by the treatment of 3μmol/L 5-aza/ 0.8% of DMSO for 24hrs at 8th passage . NKx2.5 expression was detected by relative quantitative RT-PCR 1, 2, 3 weeks after induction respectively. Results: hBMSC showed a fibroblast-like morphology before differentiation. After the treatment of 5-aza/DMSO, the former fibroblast-like cells increased in size and length in one week. The link between adjacent cells became apparent. The morphology of cell culture inclined to be unidirectioned and formed a sarcomere-like structure. RT-PCR assessment showed that the differentiated cells began to express Nkx2.5 from week 1 to week 3 of differentiation. Conclusion: These result showed that hBMSC possesses the differentiation potential to cardiomyocyte, demonstrated that 5-aza/DMSO can facilitate the cardiomyocyte differentiation in hBMSC by inducing the expression of NKx2.5.