建立绿色荧光蛋白标记的小鼠胚胎干细胞系及向心肌样细胞的分化

带有GFP基因的ESD3细胞系是一个良好的可以用于研究体内和体外细胞分化和组织产生的模型。用磷酸钙共沉淀法将质粒pEGFP-N2导入小鼠胚胎干细胞D3细胞系中 ,在荧光显微镜下以 488nm激发光检查阳性克隆 ,并进行初步扩增。经G4 18筛选后 ,机械挑取EGFP强阳性表达的克隆 ,并在丝裂霉素C处理的小鼠胚胎成纤维细胞的饲养层上 ,在无选择性压力的条件下 ,进一步扩大培养 ,获得纯化的转染细胞系。20代以后 ,转染细胞仍然表达绿色荧光蛋白。PCR检测表明 8代和 18代转染细胞均携带有GFP标志基因。对稳定表达EGFP的干细胞系进行碱性磷酸酶染色、拟胚体和畸胎瘤形成的检测 ,证明这些细胞具有干细胞的特征。经拟胚体 ,可进一步分化成具有搏动能力的心肌细胞 ,分化百分率为 30 %～ 4 0 % ,较未转染细胞 60 %～ 70 %的分化率低 ,造成低分化率机制还不清楚。这些细胞在激光共聚焦显微镜下呈绿色荧光 ,免疫组化染色显示具心肌细胞特异的cTnT分子标志。该EGFP标记的干细胞系带有可进行原位、实时检测的绿色荧光 ,可应用于细胞移植和体内分化的研究.

Establishment of Murine Embryonic Stem Cell Line Carrying Enhanced Green Fluorescence Protein and its Differentiation into Cardiomyocyte-like Cells in vitro

The availability of EGFP ES cell D3 lines provided a tractable model to study cell differentiation and tissue generation in vivo and in vitro. Plasmid pEGFP N2 was introduced into the murine embryonic stem cell D3 by standard calcium phosphate precipitation. Transfected clones were screened out under the fluorescence microscope at the 488 nm emission light in the presence of G418. Strong fluorescent EGFP clones were singly picked out and further proliferated on a feeder layer of mitomycin-C treated mouse embryonic fibroblasts. One line of EGFP ES D3 cells subcultured twenty passages and still carried the EGFP DNA without the selecting pressure. It indicated that the gene might integrate into the ES genome or still dissociated in the cytoplasm. PCR analysis for EGFP DNA showed that undifferentiated EGFP ES cells at passage 8 and 18 carried the EGFP gene. Alkaline phosphatase staining,embryoid body and teratoma formation were performed to analyze the differentiation status and potential of the EGFP ES D3 cells. The cells derived from embryoid body were able to differentiate into beating cardiomyocytes with green fluorescence clearly observable under the confocal laser scanning microscopy. 30% ～ 40% of cells from embryoid bodies were capable to differentiate into cardiomyocyte-like cells, and it appeared lower than the non-transfected ES D3 cells, which could be 60% ～ 70% under the same conditions. The mechanism was currently unknown. Immunocytochemistry staining indicated that the contracting cells were cardiomyocytes based on the presence of cardiac specific molecular marker cTnT. Results showed that the stable EGFP positive ES cell line retained the typical characteristics of ES cells and possessed the pluripotential to differentiate into beating myocytes in vitro.The EGFP transfected cells stably yielding bright green fluorescence in real time and in situ rendered it was a powerful tool in cell transplantation and tissue engineering.