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

目的构建含人cyclinD1基因全长编码区的蓝色真核荧光表达载体BFP—cyclinD1，并观察cvclinD1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-cyclinD1。转染到人乳腺癌MCF-7细胞后分为3组：实验组转染BFP—cyclinD1，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-cyclinD1，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cyclinD1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cyclinD1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。

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

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 transfeetion,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.