VEGF基因体外转染肌卫星细胞的研究

目的:寻找一种改良组织工程种子细胞的新方法,检测人血管内皮生长因子-165(VEGF165)基因转染肌卫星细胞(muscle satellite cell,MSC)的效果及目的基因的表达情况。方法:取SD大鼠的胎鼠后肢及背部肌肉,差速贴壁法接种培养MSC,观察细胞生长特性。取第2代细胞,按3:1比例用Lipofectamin2000转染试剂介导,将重组真核表达质粒pEGFP-C1-VEGF165(pEGFP为增强绿色荧光蛋白)转染MSC,转染后24～72h和传代后分别用倒置荧光显微镜观察标志基因增强绿色荧光蛋白的表达情况,计算转染效率。转染细胞爬片行VEGF165免疫组织化学染色检测VEGF165的表达。提取转染后48h的细胞总RNA,RT-PCR检测VEGF165基因的表达。结果:MSC在接种后24h开始贴壁,伸展成长梭形,原代细胞呈集簇状分布。在转染后24h即有细胞表达绿色荧光蛋白,荧光强度和表达细胞总数在72h时达高峰,传代后仍可观察到pEGFP的表达,转染后部分细胞形态发生变化。VEGF165免疫组织化学染色证实在胞质中有大量棕黄色颗粒,RT-PCR证实有与VEGF165cDNA大小相符条带出现,荧光检测转染效率约22%。结论:使用Lipofectamin2000转染试剂可有效地转染体外培养的MSC。VEGF165基因转染的MSC可表达基因产物,有望作为组织工程骨研究中的基因工程化种子细胞。

Preliminary Study on Muscle Satellite Cell Transfected with VEGF in vitro

Objective： To investigate the bone induction effectiveness and in vitro expression of SD rat muscle satellite cells after transfected by human vascular endothelial growth factor 165 （VEGF165） gene. Methods： Muscle satellite ceils were extracted from fatal SD rat and cultured in vitro by monolayer. The recombinant eukaryotic expression plasmid pEGFP-C1-VEGF165 was transfected into cells by Lipofectamin 2000. Immunohistochemiscal staining of VEGF165 was performed to detect the target protein. RT-PCR was adopted in order to find out the changes of cells after transfection. Results： Muscle satellite cells were all in the long shuttle-like shape and adhered to the disk. The expression of EGFP was appeared at 24 h after transfection, and the amount and intensity peaked at 72 h. EGFP was also seen in the second generation cells, but their expression intensity decreased. The outcome of RT-PCR and immunohistochemistry was positive. The morphology of many stem cells was modified into triangular or irregular forms under the circumstance of the secreted VEGF165. Conclusion： The VEGF165 gene can be transfected efficiently and safely into MSCs by Lipofectamin 2000. The functions of transfected cells need be further studied.