腺病毒介导的人HGF转染大鼠骨髓间充质干细胞及其作用研究

Transfection of murine mesenchymal stem cells with adenoviral vector expressing human hepatocyte growth factor and its function

目的 :评价携带HGF基因的腺病毒载体转染鼠骨髓间充质干细胞 (MSC)的效率 ,以及转染HGF基因对MSC增殖与分化的影响。方法 :采用荧光显微镜及流式细胞术检测转染效果和转染率 ;采用ELISA方法检测转染后HGF的表达情况 ;细胞增殖分析采用MTT法 ;MSC向成骨细胞分化采用碱性磷酸酶染色法。结果 :转染效率与病毒滴度 (MOI)具有量效关系 ,随着MOI的增加 ,转染率明显增高 ,当MOI =4 0 0时 ,转染率可达 99.99%。转染HGF后 ,MSC表达HGF明显增高 ,4 8h可达 12 8ng ml,随后逐渐下降并维持 2周以上 ;转染HGF对MSC的增殖以及向成骨分化没有影响。结论 :MSC是一种理想的基因载体细胞 ,可用于HGF的基因治疗。

Objective:To evaluated the efficiency of adenoviral vector expressing human hepatocyte growth factor (Ad_HGF) transfecting the murine mesenchymal stem cells (MSC) and its effect on the proliferation and differentiation of MSC. Methods:Transfection efficiency was assayed by fluorescent microscopy and flow cytometry. The expression of hepatocyte growth factor was measured with ELISA assay. MTT method was used to evaluate the proliferation of MSC. Osteogenic differentiation of MSC was assayed by akaline phosphatase staining.Results:Transfection efficiency was correlated with the multiplicity of infection (MOI) and a highest transfection efficiency (99.99%) could be achieved at MOI of 400. MSC expressed HGF at a high level (128?ng/ml) after being transfected with the Ad_HGF for 48?h. The HGF expression could maintain for at least two weeks but the level decreased with the culture time. The transfected HGF had no effect on the proliferation and differerntiation to osteocytes of MSC.Conclusions:MSC are ideal cells for cell_mediated HGF gene therapy or cell therapy. [