胶质源性神经营养因子重组腺病毒载体构建及其表达的鉴定

目的：构建携带胶质源性神经营养因子（GDNF）基因的重组腺病毒载体,鉴定其基因表达。方法：采用RT-PCR方法从人胶质瘤细胞中克隆GDNF cDNA,重组携带GDNF基因的腺病毒,鉴定其在小鼠间充质干细胞中的表达。结果：以GDNF特异性引物扩增出758bp的GDNF cDNA,经测序证实序列完全正确。GDNF基因克隆入腺病毒载体pAdCMV,细胞内重组后PCR鉴定Ad-GDNF腺病毒含有GDNF基因。重组腺病毒Ad-GDNF在小鼠间充质干细胞内能够高效表达GDNF。结论：GDNF是神经系统生长、分化和损伤后修复过程中的营养因子。以间充质干细胞作为GDNF基因治疗的供体细胞,有希望用于治疗脑缺血等继发性神经损伤以及帕金森病等神经变性疾病。GDNF基因的克隆与表达,为进一步研究GDNF对神经系统疾病的神经保护和修复作用奠定基础。

Construction and Investigation of Adenovirus Vector Carrying Glial-Derived Neurotrophic Factor Gene

Objective To construct a recombinant adenoviral vector carrying the glial-derived neurotrophic factor(GDNF) gene,and to identify GDNF expression in vitro.Methods: GDNF cDNA was amplified by RT-PCR from human glioma cells,and cloned into an adenoviral vector.The recombinant adenovirus Ad-GDNF was identified to express GDNF in the mesenchymal stem cells isolated from mouse bone marrow. Results: The 758 bp fragment of GDNF cDNA was amplified by using the specific primers,and confirmed by sequencing analysis.The recombinant adenovirus Ad-GDNF was identified by PCR to carry the GDNF gene,and can express efficiently GDNF by immunohistochemistry in the mesenchymal stem cells.Conclusions: GDNF is a neurotrophic factor in the processes of growth,differentiation and injury repair of the nervous system.As the donor cells for GDNF gene therapy,the mesenchymal stem cells may be applied in the treatment of the secondary nerve injury and the nerve degeneration diseases.The clone and expression of GDNF is the basis for the further research of the nerve protection and repair of the nervous system.