Objective To establish U251 human glioma cell line with stable overexprssion of pcDNA3. 1/connexin 43 (Cx43) re-combinant plasmid. Methods Human Cx43 primers were designed. Reverse transcriptase-polymerase chain reaction(RT-PCR) was employed to amplify targeted fragments from U251 cells with Cx43 low expression, which directly connected to pcDNA3. 1 vector after double digestion in order to construct the eukaryotic expression plasmid pcDNA3. 1/Cx43,and it was confirmed by digestion, PCR and sequencing. Lipofection method was adopted to transfer recombinant plasmid into U251 cells, the stable transfected cell line was screened by G418,and RT-PCR, Western blot were used respectively to detect the gene and protein expression level of Cx43 in U251 cells with stable transfection of pcDNA3. 1/Cx43 recombinant plasmid. Results The results of digestion, PCR and sequencing demonstrated that the pcDNA3. 1/Cx43 recombinant plasmid was successfully constructed and stable Cx43-overexprssed U251 cells were obtained by screening. Conclusion Eukaryotic expression plasmid pcDNA3. 1/Cx43 and stable Cx43-overexprssed U251 cells are constructed, which lay a experimental foundation for further study on Cx43-targeted gene therapy for human glioma.%目的 建立稳定过表达pcDNA3.1/连接蛋白43(Cx43)重组质粒的人神经胶质瘤U251细胞系.方法 设计人Cx43的引物,采用逆转录聚合酶链反应(RT-PCR)从低表达 Cx43的U251细胞中扩增出目的 片段,双酶切后定向连入pcDNA3.1载体,构建pcDNA3.1/Cx43真核表达质粒,经酶切、PCR、测序检测构建的正确性.采用脂质体转染法将重组质粒转入U251细胞,G418筛选出稳定转染的细胞系,采用RT-PCR、Western blot分别检测稳定转染pcDNA3.1/Cx43重组质粒的U251细胞中Cx43基因、蛋白的表达水平.结果 酶切、PCR及测序表明pcDNA3.1/Cx43重组质粒构建成功,筛选获得稳定过表达Cx43的U251细胞.结论 构建了pcDNA3.1/Cx43真核表达质粒及稳定过表达Cx43的U251细胞株,为下一步以Cx43为靶标进行人神经胶质瘤的基因治疗研究奠定了实验基础.
展开▼
