心脏营养素-1促进脐带间充质干细胞诱导分化为心肌样细胞时心肌α-actin、β-MHC、GATA4和 Nkx2-5基因的表达

摘要

Objective To investigate the express of α-actin, β-MHC, GATA4, Nkx2-5 gene which are effected by cardiotrophin-1 (CT-1) on differentiation of umbilical cord mesenchymal stem cells (UCMSCs) in vitro with 5-azacytidine (5-aza). Methods The UCMSCs isolated and purified from umbilical cord were divided into several groups respectively as ordinary culture medium of UCMSCs (Group A), culture medium with 0.1 nmo1/L CT-1 (Group B), ordinary culture medium including 10 μmol/L 5-aza only (Group C), culture medium combined with different concentration of CT-1 and 10 μmol/L 5-aza (Group D). The group D includes three subgroups based on the concentration of CT-1: D1 0.01 nmol/L CT-1, D2 0.1 nmo1/L CT-1, D3 1.0 nmo1/L CT-1. The fluorescent quantitation PCR was used to detect the expression levels of the α-actin, β-MHC, GATA4, Nkx2-5 gene. Results After culture for 2 or 3 days, UCMSCs adhereed to the wall, and gradually became from spherical to long fiber-like. Differentiated UCMSCs treated with 5-aza and CT-1 distinctly showed morphological characteristics of myocyte-like cells, yet spontaneous contraction of few cells wasn’t observed. There was no significant expression level of target genes in group B, C as compared with control group A. The expression levels of α-actin, GATA4, Nkx2-5 in group D were much higher than that in control group A, and each of them was sequentially highest in group D2, D1 and D3, respectively. However, there was no significant expression level of β-MHC between group D and control group A. Conclusions The phenomenon of UCMSCs differentiating into cardiomyocyte-like cells can be promoted by CT-1. The expression levels of α-actin, GATA4, Nkx2-5 are associated with the concentration of CT-1.%目的：探讨心脏营养素-1（CT-1）促进经5-氮杂胞苷（5-aza）诱导的脐带间充质干细胞（UCMSCs）分化为心肌样细胞时心肌α-actin、β-MHC、GATA4、Nkx2-5基因的表达。方法分离、纯化 UCMSCs，第4代 UCMSCs 分别以普通培养基（A 组）和含0.1 nmol/L CT-1培养基（B 组）培养；经5-aza 诱导的第4代 UCMSCs 分别以普通培养基（C 组）、含不同浓度 CT-1培养基 D 组（D1组0.01 nmol/L、D2组0.1 nmol/L、D3组1.0 nmol/L）培养。应用荧光定量 PCR 检测各基因的表达。结果 UCMSCs 培养2~3 d 后细胞贴壁，逐渐由圆形变为长纤维样；经5-aza 诱导分化后可见心肌样细胞，但未见自发性搏动。RT-PCR 检测显示 B 组和 C 组α-actin、β-MHC、GATA4和 Nkx2-5基因的表达较 A 组均无明显增加；α-actin、GATA4和 Nkx2-5基因在 D 组较 A 组表达明显增加，其中α-actin 基因在 D2组表达最高，GATA4基因在 D1组表达最高，Nkx2-5基因在 D3组表达最高，而β-MHC 基因在 D 组表达未见增加。