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目的检测胰岛素样生长因子1(IGF-1)转染肌源性干细胞效果及IGF-1基因表达情况。方法取失神经骨骼肌,分离、接种培养肌源性干细胞。取第3代细胞,用Lipofectamine介导,将重组质粒PAD-CMV-IGF-1转染肌源性干细胞。荧光显微镜观察绿色荧光蛋白的表达,酶联免疫吸附试验检测培养上清液中IGF-1的浓度变化,Western blot及实时荧光定量RT-PCR检测细胞内IGF-1及其mRNA的表达,流式细胞仪检测细胞周期。结果在转染2周内细胞表达绿色荧光蛋白。转染后上清液中分泌IGF-1浓度上升,72 h达到高峰为(32.2±5.3)ng/ml,后其浓度逐渐下降。Western blot证实有与IGF-1大小相符条带出现。实时荧光定量RT-PCR证实mRNA表达明显增高,第3天达到高峰。流式细胞检测表明转染后肌源性于细胞S期比例增加。结论PAD-CMV-IGF-1质粒可有效转染肌源性干细胞。
Objective To detect the effects of IGF-1 transfected myogenic stem cells and IGF-1 gene expression. Methods Denervated skeletal muscle was isolated, cultured and inoculated to culture myogenic stem cells. The third generation cells were obtained and transfected with recombinant plasmid PAD-CMV-IGF-1 into myogenic stem cells by Lipofectamine. Fluorescence microscopy was used to observe the expression of green fluorescent protein. The enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of IGF-1 in culture supernatant. The expression of IGF-1 and its mRNA was detected by Western blot and real-time fluorescent quantitative RT- Cytometry detects cell cycle. Results The cells expressed green fluorescent protein within 2 weeks of transfection. The concentration of secreted IGF-1 in the supernatant after transfection increased (32.2 ± 5.3) ng / ml at 72 h, and then decreased gradually. Western blot confirmed the presence of a band corresponding to the size of IGF-1. Real-time fluorescent quantitative RT-PCR confirmed mRNA expression was significantly increased, reaching the peak on the third day. Flow cytometry showed that the percentage of myogenic cells in S phase increased after transfection. Conclusion PAD-CMV-IGF-1 plasmid can effectively transfect myogenic stem cells.