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目的:探讨人脐带间质干细胞(human umbilical cord mesenchymal stem cells,hucMSCs)在体外特定条件下是否可以定向分化为心肌样细胞及干细胞标志物的表达变化。方法:采用脐带组织块贴壁培养方法分离人脐带间质干细胞,用第2代hucMSC进行体外心肌细胞诱导分化;采用RT-PCR、透射电镜等鉴定心肌细胞,流式细胞术、实时荧光定量PCR及蛋白质印迹分析干细胞相关基因及蛋白表达。结果:RT-PCR显示诱导组细胞转录心肌钙蛋白T、β-肌球蛋白重链、心房利钠多肽水平增加;在透射电镜下,诱导后细胞具有条索状肌丝结构;流式细胞仪检测未见细胞表面标志改变;而实时荧光定量PCR及蛋白质印迹显示干细胞相关基因及蛋白Nanog、Oct4、Sox2、Sall4在5-氮胞苷(5-azacytidine,5-Aza)诱导后的hucMSCs中表达显著下降。结论:人脐带间质干细胞体外经5-氮胞苷诱导可定向分化为心肌样细胞,其分化可能与干细胞相关基因及蛋白表达下调有关,为深入研究hucMSCs心肌分化机制奠定了实验基础。
OBJECTIVE: To investigate whether human umbilical cord mesenchymal stem cells (hucMSCs) can differentiate into cardiomyocytes and stem cell markers under certain conditions in vitro. Methods: Human umbilical cord mesenchymal stem cells were isolated by umbilical cord tissue culture method and induced by second passage hucMSC. Cardiomyocytes were identified by RT-PCR and transmission electron microscopy. Flow cytometry, real-time fluorescence quantitative PCR And Western blot analysis of stem cell related genes and protein expression. Results: The levels of cardiac troponin T, beta-myosin heavy chain and atrial natriuretic peptide in the induced group were increased by RT-PCR. The cells were characterized by filamentous myofilament under the transmission electron microscope. Flow cytometry Real-time PCR and Western blotting showed that the stem cell-associated genes and proteins Nanog, Oct4, Sox2 and Sall4 were expressed in hucMSCs induced by 5-azacytidine (5-Aza) Decreased significantly. CONCLUSION: Human umbilical cord mesenchymal stem cells can be induced to differentiate into cardiomyocytes in vitro by 5-azacytidine. The differentiation may be related to the down-regulation of genes and proteins related to stem cells, which lays an experimental foundation for further study of myocardial differentiation mechanism of hucMSCs.