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。
Objective ：To construct the recombinant pcI）NA3.1 -ZNF278 expression plasmid and study the func- tion 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 DH5c~. 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 MTr 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 - diphenyhetrazolium bromide assays showed that the absorbance of the pcDNA3.1 - ZNF278 - trans- fected cells was 0.739 ± 0.061, and that of the pcDNA3.1 - transfected ceils 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/G1 phase cells. Conclusion： Construction of the recombinant pcDNA3.1 - ZNF278 provides basis for ZNF278 function research. ZNF278 accelerate the cell prolif- eration and cycle of gastric cancer cell.
出处
《现代肿瘤医学》
CAS
2014年第4期795-798,共4页
Journal of Modern Oncology
关键词
ZNF278
质粒构建
细胞增殖
胃癌
ZNF278
plasmid construction
cell proliferation
gastric cancer
