NAMPT 真核表达载体构建及其对肝癌细胞增殖的影响

目的:为探索烟酰胺磷酸核糖基转移酶(NAMPT)对肝癌细胞增殖的影响,构建pc DNA3.1(+)-NAMPT真核表达载体。方法:以正常肝细胞的c DNA为模板,通过PCR扩增得到NAMPT全长序列,经酶切、连接、转化等步骤构建真核表达载体。重组质粒经酶切鉴定及测序验证后,用脂质体转染法转染肝癌细胞MHCC-97H和正常肝细胞HL-7702,免疫印迹检测NAMPT蛋白表达情况;WST实验检测过表达NAMPT后对MHCC-97H和HL-7702增殖的影响。结果:真核表达载体pc DNA3.1(+)-NAMPT构建成功,转染HL-7702和MHCC-97H细胞后,NAMPT蛋白表达水平较空白组分别升高了83%和180%;过表达NAMPT可促进细胞增殖,而抑制NAMPT活性后,细胞增殖被抑制(P0.001)。结论:成功构建了真核表达载体pc DNA3.1(+)-NAMPT,并发现NAMPT表达水平与细胞增殖密切相关,为进一步探索NAMPT表达在肝癌细胞增殖的分子作用机制和筛选新的肿瘤药物靶点奠定了基础。

Construction of NAMPT Eukaryotic Expression Vector and Its Effect on theProliferation of Hepatocarcinoma Cells

To explore the effect of NAMPT expression on the proliferation of hepatocellular carcinoma cells, pcDNA3.1(+)-NAMPT eukaryotic expression vector was constructed. Using normal liver cells cDNA as template, the NAMPT wasamplified by PCR. Eukaryotic expression vector was constructed through enzyme digestion, ligation and transformation. After identifyingthe recombinant plasmidby enzyme digestion analysis and sequencing, the recombinant plasmid was transfected into hepatocellular carcinomacell line MHCC-97H and normal liver cell line HL-7702 by liposome transfection. The NAMPT protein expression was detected byWestern Blot, and the proliferation activity of MHCC-97H and HL-7702 were measured by WST assay. FK866 was used as the inhibitorof NAMPT. pcDNA3.1(+)-NAMPT was successfully constructed in our experiment, and it was verified by restriction enzyme digestionand DNA sequencing. The NAMPT protein expression level compared with the negtive control group increased 83 %and 180 %respectively after transfecting the recombinant vector pcDNA3.1 (+)-NAMPT into the normal liver cells and hepatocellular carcinomacells. Moreover, we found that the proliferation activity was enhanced after overexpression NAMPT, and the proliferation promoting effectof NAMPT in MHCC-97H and HL-7702 were inhibited by FK866 (P<0.001). The eukaryotic expression vector pcDNA3.1(+)-NAMPT was successfully constructed, and the proliferation activity was regulated by the NAMPT expression in MHCC-97Hand HL-7702cells. These results will be a foundation for further study the roles of NAMPT in the development of Hepatocellular carcinoma.