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

The expression of α-actin, β-MHC, GATA4, Nkx2-5 gene during the process of umbilical cord mesenchymal stem cells differentiating into cardiomyocyte-like cells promoted by cardiotrophin-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。结论 CT-1可明显促进5-aza诱导的UCMSCs分化为心肌样细胞,且心肌样细胞α-actin、GATA4、Nkx2-5的基因表达与CT-1的浓度有关。

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.