胚胎干细胞SM22α-EGFP表达克隆的建立及平滑肌细胞体外发育的动态观察

Establishment of Clones of SM22α-EGFP Expressing Embryonic Stem Cells and In vitro Dynamic Tracing of Smooth Muscle Cells Development

为探讨血管发育早期血管平滑肌细胞(VSMCs)募集和增殖特点,构建了含有SM22α启动子序列和增强型绿色荧光蛋白(EGFP)编码序列的质粒,建立了平滑肌特异性蛋白SM22α启动子控制下稳定表达EGFP的胚胎干细胞株(ESCs),以研究VSMCs的发育特点.实验发现,起源于SM22α-EGFPESCs形成的胚胎小体(EBs)在第11天开启SM22α启动子并表达EGFP.此后EGFP阳性细胞持续增加,在第30天达到高峰.VSMCs多起源于EBs中细胞密集处,应用免疫荧光染色及RT-PCR观察到EGFP阳性细胞表达多种平滑肌特异性标志物.在贴壁培养的胚胎小体中VSMCs形态可分为纺锤形及上皮样的多角形,慢速视频显微摄像测得纺锤形细胞迁移速度较上皮形细胞快.以上结果表明,SM22α-EGFPESCs分化形成的EBs可以模拟体内早期胚胎血管形成过程,从形态学上获得VSMCs募集分化的证据.

A murine embryonic stem cell （ESC） line stably expressing the enhanced green fluorescent protein （EGFP） under the transcriptional control of the smooth-muscle-specific SM22α promoter to further characterize development of the vascular smooth muscle cells （VSMCs） differentiated from ESCs is established. In SM22α-EGFP expressing ESC-derived embryoid bodies（EBs）, a distinct sublineage of VSMCs could be identified by EGFP fluorescence. The SM22α promoter was switched on at day 11, and EGFP-positive cells increased gradually and reached peak at day 30. The specificity of EGFP positive cells was corroborated by RT-PCR analysis and immunostaining with antibodies against known markers for VSMCs. VSMCs were heterogeneous in their morphology in plating EBs,and could be divided into two categories： spindle-shaped or epithelioid, polygonal cells. These results suggest that SM22α-EGFP expression enables the identification of ESC -derived VSMCs by their fluorescence and morphology.