绿色荧光蛋白标记的小鼠胚胎干细胞系的建立及向心肌细胞分化

目的建立能够稳定表达绿色荧光蛋白（green fluorescent prote in,GFP）的小鼠胚胎干细胞系,并诱导其向心肌细胞分化。方法质粒pEGFP-N1脂质体复合体转染小鼠胚胎干细胞,经G418筛选后选取GFP强阳性克隆进行扩增建系。对稳定表达GFP的胚胎干细胞系进行畸胎瘤形成检测,观察其多向分化潜能。诱导GFP阳性胚胎干细胞向心肌细胞分化。免疫荧光及RT-PCR检测心肌细胞特异性标志物。结果转染后胚胎干细胞经20次传代后仍然表达GFP,裸鼠皮下接种胚胎干细胞后3050 d均可形成畸胎瘤。GFP阳性胚胎干细胞成功向心肌细胞分化。结论成功建立表达GFP的小鼠胚胎干细胞系,并可诱导其向心肌细胞分化。

Establishment of murine embryonic stem cell line carrying green fluorescent protein and its differentiation into cardiomyocytes

AIM To establish a murine embryonic stem(ES) cell line carrying green fluorescent protein(GFP) and differentiate it into cardiomyocytes.METHODS Plasmid pEGFP-N1 liposome complex was introduced into the murine ES cells and the transfected clones were filtered in the presence of G418. Strong fluorescent GFP clones were singly picked out and further proliferated.Teratoma formation was performed to analyze the differentiation status and potential of the GFP ES cells.GFP ES cells differentiated into cardiomyocytes.Immunocytochemistry staining and RT-PCR indicated that the cells derived from ES cells were cardiomyocytes based on the presence of cardiac-specific molecular marker.RESULTS One line of GFP ES cells subcultured twenty passages and still carried the GFP DNA.GFP positive ES cells were injected subcutaneously into the nude mice and tumors formed after about 30-50 days.The stable GFP ES cell line successfully differentiated into cardiomyocytes in vitro.CONCLUSION A murine ES cell line carrying GFP is established and the GFP positive ES cell line can differentiate into cardiomyocytes.