| MEK1表达载体的构建及其对肝癌细胞ERK通路活性的影响 |
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| 引用本文： | 梁容瑞,周蕊,李宗芳,李宝华,陈昆仑,王志东,姬媛媛,杨军,黄辰.MEK1表达载体的构建及其对肝癌细胞ERK通路活性的影响[J].西部医学,2013(10):1447-1450. |
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| 作者姓名： | 梁容瑞 周蕊 李宗芳 李宝华 陈昆仑 王志东 姬媛媛 杨军 黄辰 |
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| 作者单位： | [1]苏州大学附属第一医院肿瘤科,江苏苏州215006 [2]西安交通大学医学院第二附属医院普通外科干部病房,陕西西安710004 [3]生物诊断治疗国家地方联合工程研究中心,陕西西安710004 [4]西安交通大学医学院环境与疾病相关基因教育部重点实验室,陕西西安7100771 |
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| 基金项目： | 教育部“长江学者和创新团队发展计划”资助（PCSIRT：1171） |
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| 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酶切鉴定电泳奈带大小正确，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。
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| 关 键 词： | MEK1 肝癌 MHCC97H 基因表达 |
Construction and identification of recombinant expression vector contained MEK1 and its effect on ERK activity in hepatocellular carcinoma cells |
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| Institution: | LIANG Rong-rui, ZHOU Rui, LI Bao-hua, et al ( 1. Department of Oncology , The First Affiliated Hospital of Soochow University, Suzhou 215006, J iangsu, China 2. Department of General Surgery, The Second Affiliated Hospital of Xi＇an J iaotong University ,Xian 710004, China 3. National Local Joint Engineering Research Center of Biodiagnostics ＆ Biotherapy ,Xi＇an 710004, China; Key Laboratory of Environment ＆ Genes Related Diseases, College of Medicine, Xi＇an Jiaotong University, Xi＇an 710061China) |
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| Abstract: | 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. |
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| Keywords: | MEK1 Hepatocellular carcinoma MHCC97H Gene expression |
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