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中国癌症防治杂志

中国癌症防治杂志 ›› 2016, Vol. 8 ›› Issue (4): 201-206.doi: 10.3969/j.issn.1674-5671.2016.04.01

• 基础研究 •    下一篇

β-catenin过表达鼻咽癌CNE2细胞模型的构建

  

  1. 福建省科技厅转化医学重点实验室 福建医科大学教学医院 福建省肿瘤医院放射生物学及肿瘤放射治疗学研究室
  • 出版日期:2016-08-25 发布日期:2016-09-12
  • 通讯作者: 苏颖 zjsuying@hotmail.com
  • 基金资助:

    福建省自然科学基金资助项目（2014J01299）；福建省科技计划重点项目（2014Y0014）；国家临床重点专科建设项目［国卫办医函（2013）554号］

Construction of a CNE2 cellular model of nasopharyngeal carcinoma involving β-catenin overexpression

  • Online:2016-08-25 Published:2016-09-12

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摘要：

目的 构建β-catenin真核表达载体pcDNA3.1（+）/β-catenin并转染鼻咽癌CNE2细胞，检测β-catenin在CNE2细胞中的表达。方法 反转录-聚合酶链式扩增反应（reverse transcription-polymerase chain reaction，RT-PCR）扩增目的基因β-catenin，胶回收纯化后克隆至pGEM-T Easy载体，挑选阳性克隆，抽提质粒进行kpn I/xba I 双酶切和PCR鉴定，用T4 DNA连接酶将目的基因β-catenin与真核表达载体pcDNA3.1/Hygro（+）连接，构建重组的真核表达载体pcDNA3.1（+）/β-catenin，转化感受态细胞DH5α，再次抽提质粒进行PCR、kpn I/xba I双酶切和测序鉴定，然后用FuGENE HD将纯化的pcDNA3.1（+）/β-catenin转染入鼻咽癌CNE2细胞，经Hygromycin B筛选，采用实时荧光定量PCR和蛋白质印迹法（Western blot）验证转染细胞CNE2/β-catenin中β-catenin的表达。结果 PCR、双酶切及测序鉴定表明，成功构建了真核表达载体pcDNA3.1（+）/β-catenin；实时荧光定量 PCR和Western blot检测表明，与未转染细胞CNE2比较，被转染的CNE2/β-catenin细胞能够上调目的基因β-catenin的表达（P<0.01）。 结论 成功构建真核表达载体pcDNA3.1（+）/β-catenin，并能在转染的CNE2/β-catenin细胞上调目的基因β-catenin的表达。

关键词: 鼻咽肿瘤, 真核表达载体, β-catenin, 细胞模型

Abstract:

objective The eukaryotic expression plasmid pcDNA3.1（+）/β-catenin was constructed and transfected into CNE2 nasopharyngeal carcinoma cells，and β-catenin overexpression was verified in transfected cells. Methods The β-catenin gene was amplified by RT-PCR，gel-purified and cloned into pGEM-T Easy，then transformed into E.coli. Positive clones were screened by digesting extracted plasmid with Kpn I/Xba I and by analyzing with PCR. The subcloned β-catenin gene was inserted into the eukaryotic expression vector pcDNA3.1/Hygro（+） using T4 DNA ligase， resulting in the recombinant eukaryotic expression plasmid pcDNA3.1（+）/β-catenin. This construct was transformed into competent DH5α cells and positive clones were identified using PCR，Kpn I/Xba I digestion and DNA sequencing. Purified pcDNA3.1（+）/β-catenin was transfected into CNE2 cells using FuGENE HD transfection reagent，and transfectants were selected using hygromycin B. Expression of β-catenin in transfected cells was assessed using real-time fluorescence quantitative PCR and Western blotting. Results The eukaryotic expression plasmid pcDNA3.1（+）/β-catenin was constructed and confirmed by PCR，double enzyme digestion and DNA sequencing. Overexpression of β-catenin in transfected CNE2 cells was confirmed using real-time fluorescence quantitative PCR and Western blotting （P<0.01）. Conclusion  The eukaryotic expression plasmid pcDNA3.1（+）/β-catenin was constructed and can be transfected into CNE2 cells to up-regulate β-catenin. These transfected cells may provide an in vitro model for studying the role of β-catenin in nasopharyngeal carcinoma.

Key words: Nasopharyngeal neoplasm, Eukaryotic expression vector, β-catenin, Cell model