论文部分内容阅读
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 eukaryotic expression vector containing GDNF gene and analyze its expression in rat bone marrow mesenchymal stem cells. Methods Total RNA was extracted from the brain of SD rats. The GDNF gene was amplified by reverse transcription PCR. The GDNF gene was cloned into pCDNA3.1 after sequencing. The eukaryotic expression vector pCDNA3.1-GDNF was constructed. Primary culture Rat mesenchymal stem cells were transfected into mesenchymal stem cells by liposome-mediated method. RT-PCR, immunofluorescence and Western blot were used to detect the expression of GDNF Expression in mesenchymal stem cells. Results The amplified rat GDNF gene sequence was identical to the GenBank reference sequence. The GDNF gene was correctly cloned into the eukaryotic expression vector pCDNA3.1. After transfection of bone marrow mesenchymal stem cells for 4 h, GDNF mRNA and protein Correctly expressed in cells. Conclusion The pCDNA3.1-GDNF eukaryotic expression vector was successfully constructed and correctly expressed in rat mesenchymal stem cells, which laid the foundation for the further study of treatment of epilepsy with GDNF-bearing bone marrow mesenchymal stem cells.