重组质粒pIRESneo-EGFP-BDNF构建及转染骨髓间充质干细胞

Study on construction of recombinant plasmid pIRESneo-EGFP-BDNF and its expression in bone mesenchymal stem cell in mice

目的探讨重组质粒pIRESneo-EGFP-BDNF构建及转染至骨髓间充质干细胞(MSCs)制备BDNF基因工程细胞的方法。方法将pEGFP(N1)-BDNF质粒进行改造与pIRESneo相连结,构建携带BDNF的高拷贝质粒pIRESneo-EGFP-BDNF,采用电转染技术转染骨髓MSCs,经G418筛选,通过倒置荧光显微镜判断转染效率,采用Western blot方式判定转染细胞是否表达BDNF蛋白。结果经过双酶切鉴定,pIRESneo-EGFP-BDNF携带EGFP及BDNF基因,以EGFP为报告基因,质粒构建成功;通过电穿孔技术以及G418筛选,提高pIRESneo-EGFP-BDNF转染骨髓MSCs效率。结论成功制备高效表达携带BDNF基因的质粒,且转染骨髓MSCs,为进一步开展BDNF基因治疗神经系统变性疾病奠定基础。

Objective To explore the methods of recombinant plasmid plRESneoEGFPBDNF and its expression in bone mesenchymal stem cell to create BDNF engineering cell. Methods pEGFPBDNF plasmid was reconstructed into pIRESneoEGFPBDNF plasmid which was transfected to MSCs with electroproation. The transfected MSCs were bolted by G418 and were observed by blue light excited by green light under fluorescence inverted microscope. BDNF proteins were measured by Western blot in the transfected bone MSCs. Results pIRESneo-EGFP-BDNF plasmid was identified by double digestion for carrying BDNF gene and carrying EGFP gene as report gene. High efficacy of BDNF transfected to MSCs by electroporation was obtained after bolted by G418. Conclusion High-copy plasmid total expressing BDNF could be constructed and transfected to bone MSCs successfully, which played an important role in gene therapy of neurodegenerative diseases.