吉林大学学报(医学版) ›› 2013, Vol. 39 ›› Issue (2): 282-285.doi: 10.7694/jldxyxb20130220

• 基础研究 • 上一篇    下一篇

抑癌基因PTEN真核表达载体的构建及其在人舌鳞癌SCC-4细胞系中的表达

董肖婷,张斌,刘硕硕,郭婷婷,潘良朋,贾妮   

  1. 辽宁医学院附属第一医院口腔科,辽宁 锦州 121001
  • 收稿日期:2012-09-12 出版日期:2013-03-28 发布日期:2013-03-26
  • 通讯作者: 张斌(Tel:0416-4197029,E-mail:zhangbinjin@yahoo.com.cn) E-mail:zhangbinjin@yahoo.com.cn
  • 作者简介:董肖婷(1987-),女,山西省晋中市人,在读医学硕士,主要从事口腔颌面部肿瘤发生发展机制的研究。
  • 基金资助:

    辽宁省教育厅高等学校科学研究项目资助课题(L2010315)

Construction of PTEN gene eukaryotic expressing vector and its expression in tongue squamous cell carcinoma SCC-4 cell line 

DONG Xiao-ting,ZHANG Bin,LIU Shuo-shuo,GUO Ting-ting,PANLiang-peng,JIA Ni   

  1. Department of Stomatology,First Affiliated Hospital,Liaoning Medical College,Jinzhou 121001,China
  • Received:2012-09-12 Online:2013-03-28 Published:2013-03-26

摘要: [摘要] 目的:构建抑癌基因PTEN真核表达载体pEGFP-PTEN,并观察其在人舌鳞癌scc-4细胞系中的表达,为舌鳞癌的基因治疗提供理论依据。方法:提取人HeLa细胞总RNA,采用RT-PCR法获取PTEN全基因,克隆至pMD18-T载体,经EcoRⅠ和XhoⅠ双酶切后与真核表达载体pEGFP-N1连接,构建pEGFP-PTEN重组质粒。双酶切及测序鉴定后,经脂质体介导转染至体外培养的人舌鳞癌SCC-4细胞系,荧光显微镜观察绿色荧光蛋白在细胞内的表达;Western blotting法检测细胞内PTEN蛋白的表达。结果:pEGFP-PTEN重组质粒双酶切,克隆的目的基因片段约为1 200 bp,测序结果与GenBank上PTEN序列完全一致;荧光显微镜下观察到pEGFP-PTEN在SCC-4细胞系中成功表达;Western blotting结果,转染组PTEN蛋白表达强度为1.07±0.15,与空转染组(0.62±0.11)和未转染组(0.57±0.08)比较差异有统计学意义(P<0.05)。结论:成功构建pEGFP-PTEN重组真核表达载体,该载体能在人舌鳞癌SCC-4细胞系中表达。

关键词: PTEN基因, 质粒, SCC-4细胞系, 基因转染

Abstract: Abstract:Objective To construct the eukaryotic expressing vector of PTEN gene and to express the gene in SCC-4 cell line and to provide theoretical foundation for gene therapy of tongue squamous cell carcinoma.Methods  Human total length of PTEN gene was obtained from HeLa cell line by RT-PCR and was cloned into pMD18-T to analyze the sequence.The correct PTEN was inserted into pEGFP-N1 vector.pEGFP-PTEN was identified by digestion with EcoRⅠ and XhoⅠ.Scc-4 cell line was transfeted by pEGFP-PTEN and the expression of PTEN was observed by fluorescence microscope and Western blotting.Results  A 1 200 bp fragment had been cloned into pEGFP-N1 vector which was further identified by sequencing and NCBI BLAST analysis.pEGFP-PTEN was expressed successfully in SCC-4 cell line.Western blotting showed the expression of PTEN protein in pEGFP-PTEN group was 1.07±0.15,there were significant differences compared with pEGFP-N1 group(0.62±0.11) and blank control group(0.57±0.08) (P<0.05).Conclusion The eukaryotic recombinant vector pEGFP-PTEN is constructed successfully and could be overexpressed in SCC-4 cell line.

Key words: PTEN gene, plasmid construction, SCC-4 cell line, gene transfection

中图分类号: 

  • R739.86