BDNF基因转染促进骨髓间充质干细胞向神经元样细胞分化

摘要

目的:将脑源性神经营养因子(brain derived neurotrophic factor,BDNF)基因转入骨髓间充质干细胞(mesenchymal stem cells,MSC),使其作为种子细胞和基因治疗的载体将BDNF基因导入脊髓损伤组织,为探索一种更为有效的脊髓损伤治疗方法提供实验基础.方法:分离、培养、鉴定大鼠骨髓间充质干细胞；实验组应用已构建的pEGFP-N 1-BDNF进行基因转染,空质粒组用pEGFP-N1空质粒进行转染,阴性对照组只加入培养基,并进行Western印迹分析；将转染BDNF的实验组、转染空质粒组和未转染的阴性对照组细胞在体外向神经元样细胞诱导分化,比较3组细胞诱导后神经元样细胞分化率,并进行统计学分析.结果:流式细胞检测培养细胞CD90(+),CD44(+),CD34(-)、CD45(-);经western印迹检测证实实验组MSC表达BDNF-EGFP;实验组细胞向神经元样细胞分化率高于空质粒组和未转染的阴性对照组,差异具有统计学意义(p＜0.05).结论:BDNF基因转染MSC后,可以促进其向神经元样细胞分化,BDNF在MSC向神经元样细胞分化过程中具有重要作用.%Objective: To provide further foundation for the therapy for spine cord injury by transferred mesenchymal stem cells (MSC) expressing the brain-derived neurotrophic factor (BDNF) gene. Methods: Bone mesenchymal stem cells were isolated from rats and directly cultivated and expanded in vitro. Bone mesenchymal stem cells were divided into 3 groups: a pEGFP-N1-BDNF-transfected group (BDNF group), a pEGFP-N1 transfected group (vector group) and a non-transfected group. Western blot was applied to detect the expression of transgenic bone mesenchymal stem cells. The differentiation of bone mesenchymal stem cells was identified by immunofluorescence. The rates of induction of neuron-like cells among the three cell groups were compared.Results: The cell surface markers of bone mesenchymal stem cells included expression of CD90 (+), CD44 (+), CD34 (-), CD45 (-). The expression of BDNF in the transfected bone mesenchymal stem cells was demonstrated by Western blot. The positive ratio of neuron-like differentiation in the BDNF group was higher than that of both the vector group and the non-transfected group (P<0.05). Conclusion: BDNF gene plays an important role in promoting bone marrow mesenchymal stem cells to differentiate into neuron-like cells.