体外培养胚胎干细胞分化为心肌细胞特性的初步研究

Study on inducing differentiation of mouse embryonic stem cells into cardiomyocyte in vitro

目的了解干细胞在实验条件下分化发育为心肌细胞的特征表达与培养时间的关系。方法收集体外培养条件下第2、4、6、8、10、12、14、16、18和20天的胚胎干细胞，采用RT-PCR方法对其中心肌细胞特异表达基因（α／β-MHC）的表达量进行检测，并通过管家基因（GAPDH）表达量作为内对照；另外收集第0、3、5、10天培养的干细胞，检测3种管家基因（GAPDH、HGPRT、β-tublin）表达的稳定性。结果胚胎干细胞在体外特定培养条件下，第6天起α／βMHC出现，且表达量随培养时间延长逐渐增加，至14天时表达量最高，其后随着培养时间延长，表达量保持稳定；不同管家基因表达量随培养时间的变化，稳定性相差较大，其中GAPDH基因表达的波动相对稳定。结论EST实验体系分化抑制实验的周期设计为10-14天较为合理，GAPDH适合用于作为目的基因表达量检测的内对照。

Objective To study the differentiation from embeyonic stem cells into cardiomyocyte. Methods Gene α/ β-MHC ,a specific gene of cardiomyocyte and another three house-keeping genes were detected by RT-PCR from the ESCs of 0-,2-,4-,6-,8-, 10-,12-,14-,16-,18- and 20 day and ESCs of 0-,3-, 5- and 10day with specific culture condition. Results ESCs started to express α/βMHC gene from 6 day and the levels of α/βMHC increased with culture time. The levels of 14 day＇s α/βMHC were the highest. After 14 day, α/βMHC gene kept the stable levels. The levels of three housekeeping genes changed significantly with culture time, and GAPDH kept the best stability of three genes. Conclusion The cycle of the differentiation from ESCs into cardiomyocyte in vitro could be designed as 10 - 12 days and GAPDH could be selected as the fittest inner comparison.