pcDNA3.1/Cx43真核表达质粒的构建及其在人神经胶质瘤U251细胞中的稳定过表达  

Construction of eukaryotic expression plasmid pcDNA3.1/Cx43 and its stable overexpression in U251 human glioma cells

作　　者：李嘉杰[1] 闻海兵[1] 邹树峰[1] 严剑[1] 洪涛[2] 

机构地区：[1]南昌大学医学院研究生院,330000 [2]南昌大学第一附属医院神经外科,330006

出　　处：《重庆医学》2012年第9期884-887,共4页Chongqing medicine

摘　　要：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。Objective To establish U251 human glioma cell line with stable overexprssion of pcDNA3.1/connexin 43（Cx43） recombinant 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.

关 键 词：神经胶质瘤 转染 质粒 连接蛋白43 逆转录聚合酶链反应 

分 类 号：R739.4[医药卫生—肿瘤]