【摘要】 目的:构建pcDNA3.1-ZNF278重组人真核表达质粒,研究ZNF278对胃癌细胞增殖和周期的影响。方法:PCR扩增正常人胃黏膜组织中的ZNF278基因,并用EcoR I和Hind III双酶切,与酶切后的pcDNA3.1-myc-his载体连接,转化DH5α大肠杆菌,克隆筛选,提取重组质粒,酶切鉴定并测序。重组人ZNF278真核表达质粒转染胃癌细胞系SGC7901,定量PCR和Western blot技术检测ZNF278表达状况,利用MTT和细胞记数法检测细胞增殖,同时分析细胞周期变化。结果:测序证实pcDNA3.1-ZNF278真核表达质粒构建成功,并成功转染SGC7901细胞,检测确认ZNF278表达上调,转染重组pcDNA3.1-ZNF278质粒的SGC7901细胞较对照组生长加快,促进细胞周期进入S期。结论:pcDNA3.1-ZNF278重组质粒构建成功,并可促进胃癌细胞系SGC7901细胞增殖和细胞进入分裂期。更多还原

【Abstract】 Objective:To construct the recombinant pcDNA3.1-ZNF278 expression plasmid and study the function of ZNF278 in gastric cancer cell.Methods:Total RNA of normal stomach tissue was obtained.ZNF278 cDNA was amplified by PCR and digested by EcoR I and Hind III.Purified products were ligated with pcDNA3.1(+)-myc-his vector which was digested by the same enzymes,then were transformed into DH5α.The white clones were selected to be amplified and identified,the recombinant plasmid were further double-enzyme digested by EcoR I and Hind III and sequenced.Gastric cancer cell line SGC7901 was transfected with pcDNA3.1-ZNF278 or pcDNA3.1.ZNF278 over-expression SGC7901 cells were confirmed by real-time PCR and Western blot.Cell proliferation was tested by MTT and cell counting.The cell cycle was analyzed in SGC7901 cells which transfected with pcDNA3.1-ZNF278 or pcDNA3.1.Results:Recombinant pcDNA3.1-ZNF278 plasmid was proved by double enzyme-digestion and gene sequencing.Successful transfection was confirmed by real-time PCR and Western blot.3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays showed that the absorbance of the pcDNA3.1-ZNF278-transfected cells was 0.739 ± 0.061,and that of the pcDNA3.1-transfected cells was 0.5092 ± 0.027.The cell growth curve showed that the in vitro tumor cell growth was significantly promoted in the cells transfected with pcDNA3.1-ZNF278 plasmid as compared to control cells.The over-expression of ZNF278 plasmid significantly increased the percentage of the S phase cells and decreased the percentage of the G0/G1phase cells.Conclusion:Construction of the recombinant pcDNA3.1-ZNF278 provides basis for ZNF278 function research.ZNF278 accelerate the cell proliferation and cycle of gastric cancer cell.更多还原