胚胎干细胞来源的拟胚体分化早期血管平滑肌标志物的表达时相

EXPRESSION OF VASCULAR SMOOTH MUSCLE CELL MARKERS DURING EARLY STAGE OF EMBRYONIC STEM CELL-DERIVED EMBRYOID BODIES DIFFERENTIATION

目的:研究胚胎血管发育早期SMα-actin、SM22α、myocardin、平滑肌肌球蛋白重链(SMMHC)的表达规律,并初步探讨在此阶段血小板源性生长因子-BB(PDGF-BB)对血管平滑肌细胞(VSMCs)分化的影响。方法:采用转染平滑肌特异性蛋白SM22α启动子控制下表达增强型绿色荧光蛋白(GFP)报告基因载体的胚胎干细胞制备拟胚体(EBs),用免疫荧光染色、RT-PCR、Western blot分析SMα-actin、SM22α、myocardin、SMMHC的表达时相;然后分别用0μmol/L(对照组)、10μmol/L、50μmol/L AG1296(血小板源性生长因子受体抑制剂)处理EBs,观察三组SMα-actin、SM22α、myocardin、SMMHC在基因及蛋白水平上的表达变化。结果:胚胎血管发育早期SMα-actin、myocardin、SM22α、SMMHC分别在EBs第0(胚胎干细胞)、8、11、13d开始有表达。AG1296三种浓度处理后SMα-actin、myocardin、SM22α、SMMHC蛋白表达及myocardin、SM22α和SMMHC mRNA表达均无明显差异。结论:EBs发育过程中存在着自发的VSMCs分化,SMα-actin表达最早,依次为myocardin、SM22α、SMMHC;PDGF-BB对EBs分化早期VSMCs标志物表达的调控可能不是必要的。

Aim： To observe expression regularity of SMα-actin, SM22α, myocardin and SMMHC during early embryonic vascular development, and to initially investigate the differentiation effect of platelet derived growth factor-BB（PDGF-BB） on vascular smooth muscle cells （VSMCs） during that period. Methods： Murine embryonic stem call line expressing the enhanced green fluorescent protein（GFP） under the transcriptional control of the smooth-muscle-specific SM22α promoter was used to make embryoid bodies, and to analyze the expression regularity of SMα-actin, SM22α, myocardin and SMMHC by immunofluorescence stainings, RT-PCR and Western blot. Then AG1296 （PDGF receptor inhibitor） 0μmol/L（control group）, 10 μmol/L and 50 μmol/L were used to treat EBs respectively in order to analyze the differences of SMα-aetin, SM22α, myocardin and SMMHC at gene and protein levels among the three groups. Results： SMα-actin, myocardin, SM22α and SMMHC expression in EBs were found to begin at day 0 （ESCs）, 8,11,13 respectively during early embryonic vascular development.There were no clear differences in SMα-actin, SM22α, myocardin and SMMHC protein expression and SM22α, myocardin and SMMHC mRNA level among the three groups of different concentrations of AG1296. ：Conclusion： A spontaneous VSMCs differentiation occurs during EBs development,SMα-actin is the first to be detected,the following are myocardin, SM22α and SMMHC. PDGF-BB may not be indispensable for the regulation of expression of VSMCs markers during early EBs differentiation.