MyoD基因诱导骨髓间充质干细胞分化为成肌细胞的实验研究

Experimental studies on the differentiation of bone marrow mesenchymal stem cells into myoblasts induced by MyoD gene in vitro

目的 探讨MyoD基因诱导骨髓间充质干细胞 (MSCs)分化为成肌细胞的可能性。方法 利用脂质体转染方法 ,将真核表达的双顺反子质粒载体pIRES2 EGFP MyoD转入MSCs中 ,G418筛选 ;用RT PCR检测MyoD的表达 ,扩增产物纯化测序鉴定 ;荧光显微镜和激光共聚焦观察报告基因产物 ;免疫组化检测MyoD、myogenin、myosin、myoglobin、desmin的表达 ;电镜观察转染前后细胞超微结构的变化。结果 RT PCR可检测出转染后细胞表达MyoD ,扩增产物纯化测序与GeneBank比较完全一致 ;荧光显微镜和激光共聚焦观察均可见绿色荧光 ;免疫组化检测MyoD、myogenin、myosin、myoglobin、desmin表达均为阳性 ;转染后的MSCs观察表现为较为成熟细胞的形态学特点 ,且胞浆中有丝状物结构。结论 MyoD基因可成功诱导体外培养的骨髓MSCs分化为成肌细胞 。

Objective To explore the possibility of MyoD gene inducing bone marrow mesenchymal stem cells (MSCs) to differentiate into myoblasts in vitro Methods The eukaryotic expression bicistron plasmid vector pIRES2 EGFP MyoD was transfected into MSCs with lipotransfection method followed by G418 selection The expression of MyoD in the transfectants was detected with RT PCR and the amplified, purified product was identified with sequence analysis The reporter gene (Enhance green fluorescent protein, EGFP) was observed in the transfected cells under the fluorescent and the laser cofocal microscopes Immunohistochemical methods were employed to study the expressions of MyoD, myogenin, myosin, myoglobin and desmin The ultrastructure changes of cells before and after transfection were observed with electron microscopy Results The expression of MyoD was detected in the transfected MSCs with RT PCR and the amplified, purifed product was in same sequence with that from Genbank Green fluorescence was observed in the transfectants under the fluorescent and the laser cofocal microscopes Immunohistochemical methods indicated the expressions of MyoD, myogenin, myosin, myoglobin and desmin respectively in the transfected cells The transfected cells were of characteristic of filaments in their cytoplasm Conclusion MyoD gene could induce cultured MSCs to differentiate into myoblasts, thus providing an experimental foundation for trauma repair