含GDNF基因真核载体的构建及其在大鼠骨髓间充质干细胞中的表达

Construction of eukaryotic expression vector for GDNF and its expression in rat bone marrow mesenchymal stem cells

目的构建含GDNF基因真核表达载体,并分析其在大鼠骨髓间充质干细胞中的表达。方法从SD大鼠脑中提取总RNA,采用逆转录PCR法扩增GDNF基因,测序鉴定后将GDNF基因克隆入p CDNA3.1中,构建真核表达载体p CDNA3.1-GDNF;原代培养大鼠间充质干细胞,以脂质体介导法将构建好的真核表达载体p CDNA3.1-GDNF转染至间充质干细胞;RT-PCR、细胞免疫荧光、Western blot检测GDNF在间充质干细胞中的表达。结果扩增的大鼠GDNF基因序列与Gen Bbank的参考序列完全一致,GDNF基因已经正确克隆到真核表达载体p CDNA3.1中;转染骨髓间充质干细胞4 h后,GDNF的mRNA和蛋白能在细胞中正确表达。结论 p CDNA3.1-GDNF真核表达载体构建成功,并能在大鼠间充质干细胞中正确表达,这为下一步研究携带GDNF的骨髓间充质干细胞治疗癫痫奠定了实验基础。

Objective To construct the eukaryotic expression vector of GDNF and analyze their expression in rat bone marrow mesenchymal stem cells （BMSCs）. Methods The total RNA was extracted from the brain of SD rat, using RT-PCR amplification of GDNF gene after sequenclng,the GDNF gene was cloned into pCDNA3.1 and the eukaryotic expression vector of pCDNA3.1-GDNF was successfully constructed. Primary cultured rat mesenchymal stem cells, the recombinant plasmid of pCDNA3.1-GDNF was transfected into BMSCs via Lipofectamine. Using RT-PCR, Western blot and immunofluorescence assay to examine the expression of the GDNF. Results Amplification of rat GDNF gene sequences compared to the GenB bank published reference sequence was exactly the same. results of double enzyme digestion proved that GDNF gene has been correctly cloned into the eukaryotic expression vector pCDNA3.1, GDNF successful expression in BMSCs at the mRNA and protein levels 4 hours after transfection with the recombinant plasmid. Conclusion pCDNA3.1-GDNF eukaryotic expression vector was successfully constructed, and it expressed correctly in mesenchymal stern cells of SD rat. This laid the foundation for the further cell transplantation.