hFXYD6真核表达载体构建及对人肝癌细胞系SMMC7721细胞的生物学作用的影响  

Construction of pcDNA3.1-h FXYD6 eukaryotic expression vector and effect on biological behavior of hepatocarcinoma SMMC7721 cells

作　　者：陈雄飞[1] 张执全[1] 袁俊建[1] 郭忠健[2] 郭尧 李凤山[1] 刘汝海[1] 

机构地区：[1]沧州市中心医院普外一科,河北沧州061001 [2]沧州市中心医院病理科,河北沧州061001 [3]沧州市中心医院中心实验室,河北沧州061001

出　　处：《生物医学工程与临床》2017年第6期660-663,671,共5页Biomedical Engineering and Clinical Medicine

摘　　要：目的构建人肿瘤相关蛋白FXYD6真核表达载体pc DNA3.1-h FXYD6,研究其对人肝癌细胞系SMMC7721细胞增殖与侵袭的影响。方法扩增h FXYD6 c DNA,将其插入克隆质粒载体p MD18 T Simple,将限制性内切酶酶切后的目的片段插入以限制性内切酶切后的表达质粒载体pc DNATM 3.1/myc-His(-)B中,经聚合酶链反应(PCR)电泳和测序证实后转染至人肝癌细胞系SMMC7721细胞中,同时设pc DNA3.1空载体转染组和pc DNA3.1-h FXYD6载体转染组,比较两组细胞的增殖与侵袭情况。结果成功构建了pc DNA3.1-h FXYD6真核表达载体。转染真核质粒pc DNA3.1-h FXYD6的SMMC7721细胞,经培养48 h,电泳图显示用h FXYD6单克隆抗体识别的特异性蛋白条带。pc DNA3.1-h FXYD6表达载体组细胞较pc DNA3.1空载体转染组细胞增殖速度明显增快。pc DNA3.1空载体转染组、pc DNA3.1-h FXYD6表达载体组细胞穿透Boyden趋化小室滤膜的细胞数分别为(109.9±7.8)个、(167.6±9.3)个,两组差异有显著统计学意义(P<0.001)。结论成功构建的pc DNA3.1-h FXYD6真核质粒能在肝癌细胞系SMMC7721细胞中表达目的蛋白,h FXYD6可促进肝癌细胞增殖与侵袭,这为研究h FXYD6的具体作用机制奠定了基础。Objective To construct human tumor associated protein FXYD6 eukaryotic expression plasmid pc DNA3.1-h FXYD6, and explore its effect on proliferation and invasion of human hepatic carcinoma SMMC7721 cells. Methods The h FXYD6 c DNA was amplified and cloned into vector p MD18-T Simple. The recombinant was digested by restriction endonu-clease, and the interest fragment was inserted into pc DNATM3.1/myc-His（-） B expression vector digested by the same restriction endonuclease. Confirmed by polymerase chain reaction（PCR） electrophoresis and sequence analysis, the verified recombinant was transected in human hepatocarcinoma SMMC7721 cells. The pc DNA3.1 empty vector transfected group and pc DNA3.1-h FXYD6 transfected group were set, and compared proliferation and invasion between 2 groups. Results The eukaryocyte expression vector pc DNA3.1-h FXYD6 was constructed. The eukaryotic plasmid pc DNA3.1-h FXYD6 was transfected into SMMC7721 cells, the electrophoregram showed specific protein band identified with h FXYD6 monoclonal antibody after incu-bation for 48-hour. The cell proliferation rate of pc DNA3.1-h FXYD6 transfected group was faster than that of pc DNA3.1 emp-ty vector transfected group. The cell number of penetrating Boyden chemotaxis in pc DNA3.1 empty vector transfected group and pc DNA3.1-h FXYD6 transfected group was（109.9 ± 7.8） and（167.6 ± 9.3）, respectively, and the difference was statistically significant（P〈0.001）. Conclusion It is demonstrated that the constructed eukaryotic expression plasmid pc DNA3.1-h FXYD6 could express the target protein in human hepatic carcinoma SMMC7721 cells, and h FXYD6 could promote the proliferation and invasion of hepatocarcinoma SMMC7721 cells, which lay the foundation for the study of specific mechanism of h FXYD6.

关 键 词：hFXYD6 真核表达 载体转染 肝肿瘤 SMMC7721细胞 生物学效应 

分 类 号：R735.7[医药卫生—肿瘤]