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中国癌症防治杂志

中国癌症防治杂志 ›› 2012, Vol. 4 ›› Issue (2): 174-178.doi: 10.3969/j.issn.1674-5671.2012.02.19

• 基础研究 • 上一篇    下一篇

重组质粒BFP-cyclin D1的构建及表达对乳腺癌MCF-7细胞增殖和迁移能力的影响

  

  1. 广西医科大学第一附属医院病理科；广西医科大学药学院；广西医科大学医学实验中心
  • 出版日期:2012-06-25 发布日期:2012-07-03
  • 通讯作者: 徐 恒 E-mail:heng6888@yahoo.com
  • 基金资助:

    国家自然科学基金资助项目（30672363）；广西自然科学基金资助项目（2010GXNSFC013017）；广西医科大学研究生创新课题（2010105981001M227）

Construction and expression of the recombinant plasmid BFP-cyclin D1 and its influence on proliferation and migration in breast cancer cells MCF-7

  • Online:2012-06-25 Published:2012-07-03

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被引次数

7

摘要： 目的 构建含人cyclin D1基因全长编码区的蓝色真核荧光表达载体BFP-cyclin D1，并观察cyclin D1的表达对MCF-7增殖和迁移能力的影响。方法 以人乳腺癌MCF-7细胞总RNA为模板，经PCR扩增和酶切后与pEBFP-N1质粒连接，得到重组质粒BFP-cyclin D1。转染到人乳腺癌MCF-7细胞后分为3组：实验组转染BFP-cyclin D1，阴性对照组转染pEBFP-N1；空白对照组为MCF-7，以荧光定量PCR检测基因表达；MTT实验检测细胞生长速度；细胞划痕实验检测细胞迁移能力。结果 成功构建了BFP-cyclin D1，并在人乳腺癌MCF-7细胞中呈高表达，荧光定量PCR检测示实验组cyclin D1在转染后12h开始增高，48h达到高峰并持续高表达，与激光共聚焦显微镜观察实验组蓝色荧光表达量的趋势一致；MTT实验证实实验组细胞增殖速度（48、72、96h）显著高于对照组（P<0.01）；细胞迁移实验表明实验组细胞迁移能力显著高于对照组细胞（P<0.01）。结论 cyclin D1的高表达促进了人乳腺癌MCF-7细胞的增殖和迁移，这可能与其缩短细胞周期、促进DNA合成和复制功能有关。 

关键词: 乳腺癌, cyclin D1, MCF-7, 细胞增殖、迁移

Abstract: Objective To construct the eukaryotic expression plasmid BFP-cyclin D1 and investigate the effects of cyclin D1 on the proliferation and migration of MCF-7 cells. Methods The cyclin D1 gene was amplified by RT-PCR from total RNA of MCF-7 cells， digested with EcoR I and Sal I， and inserted into the eukaryotic florescence expression vector pEBFP-N1.The resulting recombinant plasmid BFP-cyclin D1 was transfected into MCF-7 cells.In parallel，one negative control group was transfected with pEBFP-N1 and another with transfection reagent without any DNA.Following transfection，cell proliferation was detected by MTT assay，cell migration by cell scratch assay，and mRNA expression by quantitative real-time PCR. Results The eukaryotic expression plasmid BFP-cyclin D1 was successfully constructed and high expression of cyclin D1 was detected in MCF-7 cells.Cell proliferation and migration were significantly higher in the cells transfected with BFP-cyclin D1 than in the two control groups（P<0.01）. Conclusions High expression of cyclin D1 can accelerate proliferation and migration of MCF-7 cells，perhaps by shortening the cell cycle and promoting DNA replication.

Key words: Breast cancer, Cyclin D1, MCF-7, MTT, Cell proliferation and migration