摘要
目的构建pcDNA3.1(+)-MEK1真核表达载体,体外转染人肝癌MHCC97H细胞,观察MEK1在肝癌细胞中的表达及其对ERK通路活性的影响。方法应用RT-PCR扩增出MEK1基因插入pcDNA3.1(+)中,形成重组载体pcDNA3.1(+)-MEK1。经测序确认后,利用LipofectamineTM 2000将重组载体转染MHCC97H肝癌细胞,使用含G418的培养基进行筛选;将肝癌细胞分为不转染组、转染空载体组和转染重组载体组,运用Western-blot方法检测MEK1、p-MEK1、ERK1/2、p-ERK1/2在各组细胞中的表达并绘制各组细胞的生长曲线对比其增殖能力。结果重组载体pcDNA3.1(+)-MEK1酶切鉴定电泳条带大小正确,测序结果经Blast比对与人MEK1基因开放性读码框100%吻合;重组载体载体转染肝癌细胞后MEK1表达水平较不转染及转染空载体载体组细胞显著升高,且磷酸化的MEK1及ERK1/2水平也明显升高,转染重组载体载体的肝癌细胞增殖能力得到显著增强。结论成功构建了真核表达载体pcDNA3.1(+)-MEK1,过表达MEK1蛋白可提高肝癌细胞MAPK/ERK通路活性。
Objective To construct the expression vector of pcDNA3.1（＋）-MEK1 and observe its effect on ERK activity human hepatocellular carcinoma cells MHCC97H. Methods The full length open reading frame of human MEK1 （MAP2K1） gene was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1 （＋）. After identified by enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into human hepatocellular carcinoma cells using liposomes 2000, and the expression of MEK1, p-MEK1, ERK1/2 and p-ERK1/2 were detected by Western- blot. Growth curve assay was executed to evaluate the proliferation of cells post-transfection. Results Plasmid was di- gested, and the amplified fragments had 100% homology with the human MEK1 gene published in the Gene bank. Cells cultured in medium contained G418 with a concentration of 500mg/ml for 21 days could be indentified into transfected cells or not. The Western blot demonstrated a high expression of MEK1, p-MEK1 and p-ERK1/2 in MHCC97H cells af- ter trasfected by recombinant plasmid. A pro-proliferative effect due to recombinant plasmid was detected. Conclusion The eukaryotic expression vector pcDNA3.1（＋）-MEK1 was constructed successfully and the MEK1 protein can be ex- pressed successfully in human hepatocellular carcinoma cells MHCC97H, therefor induce a highly activation of ERK sig- naling in HCC cells.
出处
《西部医学》
2013年第10期1447-1450,共4页
Medical Journal of West China
基金
教育部"长江学者和创新团队发展计划"资助(PCSIRT:1171)
