摘要
目的:构建肌细胞增强因子2A(MEF2A)基因真核表达重组质粒,并转染乳腺癌细胞株观察其对细胞增殖能力的影响。方法:实时定量聚合酶链反应(RT-QPCR)检测MCF-7、BT474及MDA-MB-231等3种乳腺癌细胞中MEF2A mRNA的表达。采用全基因合成法合成MEF2A编码区序列,克隆入pcDNA3.1(+)-HA载体,构建重组质粒pcDNA3.1-MEF2A-HA。重组质粒转染乳腺癌细胞,采用Western blot及RT-QPCR检测MEF2A的表达效率。细胞计数实验检测细胞的增殖能力。结果:3株乳腺癌细胞中,MDA-MB-231细胞中MEF2A mRNA表达水平较低;其基因测序结果显示与预期片段大小一致,表明MEF2A重组质粒构建成功。将MEF2A重组质粒瞬时转染MDA-MB-231细胞后,MEF2A表达质粒组与对照组比较,其MEF2A表达升高,细胞增殖能力增强。结论:成功构建MEF2A过表达重组质粒,在MDA-MB-231中过表达MEF2A促进细胞的增殖能力。
Objective: To construct eukaryotic expression plasmid of pcDNA3.1-MEF2A and examine its effect on the proliferation of breast cancer cell line. Methods: The MEF2A mRNA levels were detected in MCF-7, BT474 and MDA-MB-231 cells, respectively by real-time quantitative PCR(RT-QPCR). The full-length coding sequence of MEF2A was amplified by standard RT-QPCR. The amplified MEF2A was cloned into pcDNA3.1 vector. The eukaryotic expression plasmid pcDNA3.1-MEF2A-HA was transfected into breast cancer cells. RT-QPCR and western blot were performed to measure the expression level of MEF2A. Cell counting was performed to evaluate the effects of MEF2A expression on cell proliferation. Results: The MEF2A mRNA expression level was low in MDA-MB-231 cells. Western blot and RT-QPCR indicated that transfection of MEF2A expression vector into MDA-MB-231 cells resulted in increased mRNA/protein levels of MEF2A. The forced MEF2A expression significantly enhanced cell proliferation of MDA-MB-231. Conclusion: The eukaryotic expression plasmid of pcDNA3.1-MEF2A-HA is constructed successfully. Over-expression of MEF2A enhances proliferation ability of MDA-MB-231 cells.
出处
《天津医科大学学报》
2014年第6期429-432,共4页
Journal of Tianjin Medical University
关键词
肌细胞增强因子2A
乳腺癌
质粒
增殖
myocyte enhancer factor 2A
breast cancer
plasmid
proliferation