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目的选择从人脐带血中分离出的间充质干细胞(UCB1-MSCs),通过5-氮胞苷(5-aza)进行诱导,对其体外向心肌细胞转分化的能力做初步探讨。方法培养第11代 hMSCs,加入6、9、12 μmol/L 的5-aza 分别作用24h 和48h,相差显微镜观察细胞形态直到3周后实验结束。以未经处理的细胞为对照组。采用 RT-PCR 检测心肌转录因子 MEF2C、GATA4、Nkx2.5和心肌特异性基因 MLC-2v、MLC-2a、Connexin 43及α-actinin 的表达;免疫荧光染色检测 Connexin 43和α-actinin 的表达,以共聚焦显微镜成像。结果经5-aza 诱导后观察3周未见细胞的自发搏动。诱导前后的 hMSC 均可不同程度的表达 MEF2C、Connexin 43及α-actinin。在12 μmol/L 水平诱导24h 和6、9、12 μmol/L 水平诱导48h 共4个组中可见 MLC-2a 的表达。GATA4、Nkx 2.5和 MLC-2v 在诱导前后均未见表达。免疫荧光染色显示诱导前后 hMSCs 的胞浆内存在散在排列无序的α-actinin 和 Connexin 43荧光,而始终未见肌小节结构。结论经5-aza 诱导 UCB 来源 hMSCs 在基因水平可以表达部分心肌特异性基因,但是在诱导后的一个月时间内未见相应的心肌特异性结构出现,此类细胞向心肌细胞转分化的能力需再证实。
Objective To select the mesenchymal stem cells (UCB1-MSCs) isolated from human umbilical cord blood and induce them by 5-azacytidine (5-aza) to investigate their ability to transdifferentiate into cardiomyocytes in vitro. Methods The 11th generation hMSCs were cultured and treated with 6, 9, 12 μmol / L 5-aza for 24h and 48h respectively. The morphology of the cells was observed by phase contrast microscopy until the end of 3 weeks. The untreated cells as a control group. The expression of myocardial transcription factors MEF2C, GATA4, Nkx2.5 and myocardial specific genes MLC-2v, MLC-2a, Connexin 43 and α-actinin were detected by RT-PCR. The expressions of Connexin 43 and α-actinin were detected by immunofluorescence staining. Confocal microscopy imaging. Results No spontaneous beating of cells was observed after 3-week induction with 5-aza. Before and after induction of hMSC can express MEF2C, Connexin 43 and α-actinin to varying degrees. The expression of MLC-2a was observed in 4 groups induced by 12 μmol / L for 24 h and 6, 9, 12 μmol / L for 48 h. GATA4, Nkx 2.5 and MLC-2v were not expressed before and after induction. Immunofluorescence staining showed that there was disorder of α-actinin and Connexin 43 fluorescence in the cytoplasm of hMSCs before and after induction, but there was no muscle structure. Conclusion 5-aza-induced hBMSCs derived from UCB can express some myocardial specific genes at the gene level, but no specific myocardial structure appears within one month after induction. The ability of these cells to transdifferentiate into cardiomyocytes Confirm again.