天津医药 ›› 2018, Vol. 46 ›› Issue (2): 126-131.doi: 10.11958/20171057

• 细胞与分子生物学 • 上一篇    下一篇

慢病毒载体介导的bFGF基因转染兔BMSCs的实验研究

张槐1 , 彭吾训2△, 刘钢2 , 张健2 , 张飞1 , 王健波1 , 赵胤1 , 李青2   

  1. 1贵州医科大学 (邮编550004); 2贵州医科大学附属医院骨科
  • 收稿日期:2017-10-10 修回日期:2017-12-18 出版日期:2018-02-15 发布日期:2018-02-15
  • 通讯作者: 张槐 E-mail:986693631@qq.com
  • 基金资助:
    贵阳市科技局基金项目

Experimental study of lentiviral vector - mediated bFGF gene transfection on rabbit BMSCs

ZHANG Huai 1 , PENG Wu-xun2△, LIU Gang2 , ZHANG Jian2 , ZHANG Fei 1 , WANG Jian-bo1 , ZHAO Yin1 , LI Qing2   

  • Received:2017-10-10 Revised:2017-12-18 Published:2018-02-15 Online:2018-02-15
  • Contact: Huai ZHANG E-mail:986693631@qq.com

摘要: 摘要: 目的 观察在体外培养条件下, 慢病毒载体介导的碱性成纤维细胞生长因子 (bFGF) 基因转染对兔骨髓基质干细胞 (BMSCs) 生物学特性的影响。方法 采用密度梯度离心法及贴壁筛选法获取 BMSCs; 利用慢病毒载体将 bFGF 基因转染到 BMSCs 中, 根据转染条件分为 bFGF 转染组、 空病毒组、 未转染组, 转染后分别对 3 组细胞的形态 bFGF mRNA 及蛋白表达、 细胞增殖情况、 细胞周期及碱性磷酸酶 (ALP) 活性进行观察。结果 采用密度梯度离心法和贴壁筛选法, 成功获得高纯度的 BMSCs。载有 bFGF 基因的慢病毒转染 BMSCs 后, 细胞形态无明显变化, 而 bFGF mRNA 与蛋白表达均明显增强、 细胞增殖曲线上移、 增殖期细胞比例增大、 ALP 活性明显增高, 与空病毒组及未转染组比较, 差异均有统计学意义 (P<0.05)。结论 利用慢病毒载体将兔 bFGF 基因导入体外培养的 BMSCs, 目的基因获得稳定表达, 并且自身过表达的bFGF 可以促进BMSCs 的增殖与成骨分化。

关键词: 关键词: 干细胞, 成纤维细胞生长因子2, 遗传载体, 转染, 细胞增殖, 骨髓基质干细胞, 慢病毒载体

Abstract: Abstract: Objective To observe the effect of lentiviral vector-mediated basic fibroblast growth factor (bFGF) gene transfection on the biological characteristics of rabbit bone marrow stromal cells (BMSCs) under in vitro culture conditions. Methods BMSCs were obtained by density gradient centrifugation and adherence screening. The bFGF gene was transfected into BMSCs by lentiviral vector and divided into bFGF transfection group, empty virus group and untransfected group according to the transfection conditions. After transfection, the morphology, expressions of bFGF mRNA and protein, cell proliferation, cell cycle and alkaline phosphatase (ALP) activity were observed in three groups of cells. Results High density BMSCs were successfully obtained by density gradient centrifugation and adherence screening. After transfection of BMSCs with bFGF gene, the cell morphology showed no significant changes, while the expressions of bFGF mRNA and protein were significantly increased, the cell proliferation curve shifted upward, the proportion of proliferating cells increased, and the activity of ALP was significantly enhanced. There were significant differences between three groups (P < 0.05). Conclusion The rabbit bFGF gene is successfully introduced into the BMSCs cultured in vitro by lentiviral vector, and the target gene is stably expressed. The expression of bFGF can promote the proliferation and osteogenic differentiation of BMSCs.

Key words: Key words:stem cells, fibroblast growth factor 2, genetic vectors, transfection, cell proliferation, bone marrow stromal cells, lentiviral vector