| 王姣姣 朱剑军 张卉 任婷婷 金明朋 邢金良 吴有盛.NAMPT 真核表达载体构建及其对肝癌细胞增殖的影响[J].,2016,16(13):2401-2404 |
| NAMPT 真核表达载体构建及其对肝癌细胞增殖的影响 |
| Construction of NAMPT Eukaryotic Expression Vector and Its Effect on theProliferation of Hepatocarcinoma Cells |
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| DOI： |
| 中文关键词: 尼克酰胺磷酸核糖转移酶(NAMPT) 细胞增殖 肿瘤代谢 载体构建 |
| 英文关键词: Nicotinamide phosphoribosyltransferase (NAMPT) Cell proliferation Tumor metabolism Eukaryotic expression vector
construction |
| 基金项目:国家自然科学基金项目（81572727） |
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| 中文摘要: |
| 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pcDNA3.1(+)-NAMPT 真核表达载体。方法：
以正常肝细胞的cDNA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pcDNA3.1(+)-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(P<0.001)。结论：成功构建了真核表达载体pcDNA3.1(+)-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。 |
| 英文摘要: |
| 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 was
amplified by PCR. Eukaryotic expression vector was constructed through enzyme digestion, ligation and transformation. After identifying
the recombinant plasmidby enzyme digestion analysis and sequencing, the recombinant plasmid was transfected into hepatocellular carcinoma
cell line MHCC-97H and normal liver cell line HL-7702 by liposome transfection. The NAMPT protein expression was detected by
Western Blot, and the proliferation activity of MHCC-97H and HL-7702 were measured by WST assay. FK866 was used as the inhibitor
of NAMPT. pcDNA3.1(+)-NAMPT was successfully constructed in our experiment, and it was verified by restriction enzyme digestion
and 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 carcinoma
cells. Moreover, we found that the proliferation activity was enhanced after overexpression NAMPT, and the proliferation promoting effect
of 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-97H
and HL-7702cells. These results will be a foundation for further study the roles of NAMPT in the development of Hepatocellular carcinoma. |
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