| 靶向Nrf2基因microRNA表达载体构建及初筛 |
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| 引用本文： | 李煌元,汤章彬,吴思英,林炜,王章敬. 靶向Nrf2基因microRNA表达载体构建及初筛[J]. 中国公共卫生, 2011, 27(9) |
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| 作者姓名： | 李煌元 汤章彬 吴思英 林炜 王章敬 |
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| 作者单位： | 1. 福建医科大学公共卫生学院环境与健康研究所职业与环境卫生学系,福建福州,350004
2. 福建医科大学公共卫生学院流行病与卫生统计学系 |
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| 基金项目： | 国家自然科学基金，福建省环境与健康重点学科基金，福建省教育厅 |
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| 摘 要： | 目的 利用真核表达载体pcDNATM6.2-GW/EmGFP miR载体构建针对人Nrf2基因的微小RNA( microRNA)真核表达载体,为研究Nrf2基因在化学物毒作用提供参考依据.方法 设计特异性针对Nrf2基因的寡核苷酸序列,构建重组载体并命名为pcDNA-Nrf2-A、pcDNA-Nrf2-B、pcDNA-Nrf2-C、pcDNA-Nrf2-D,转染人乳腺癌MCF-7细胞；通过荧光显微镜观察绿色荧光监测转染效率,转染48 h后用荧光定量逆转录-聚合酶链反应(RT-PCR)和免疫细胞化学检测瞬时转染细胞Nrf2基因mRNA和蛋白的表达变化观察沉默作用来初筛有效的重组载体.结果 成功构建miRNA真核表达载体pcDNA-Nrf2;转染效率约为20% ～40%；瞬时转染重组质粒48 h后,与空白对照和阴性对照质粒比较,pcDNA-Nrf2-A Nrf2 mRNA表达差异无统计学意义(P＞0.05)；pcDNA-Nrf2-B、pcDNA-Nrf2-C、pcDNA-Nrf2-D mRNA表达降低,差异有统计学意义(P ＜0.001),pcDNA-Nrf2-C抑制Nrf2基因mRNA表达强于pcDNA-Nrf2-D(P ＜0.05).结论 成功构建了Nrf2的microRNA表达载体pcDNA-Nrf2-C,可作为进一步研究Nrf2基因功能的工具.
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| 关 键 词： | Nrf2 微小RNA 真核表达载体 人乳腺癌细胞株MCF-7 |
Construction and primary selection of microRNA expression vector directed against Nrf2 |
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| LI Huang-yuan,TANG Zhang-bin,WU Si-ying,et al.. Construction and primary selection of microRNA expression vector directed against Nrf2[J]. Chinese Journal of Public Health, 2011, 27(9) |
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| Authors: | LI Huang-yuan TANG Zhang-bin WU Si-ying et al. |
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| Affiliation: | LI Huang-yuan,TANG Zhang-bin,WU Si-ying,et al.Department of Occupational and Environmental Health,Institute of Environment and Occupation Health,School of Public Health,Fujian Medical University(Fuzhou 350004,China) |
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| Abstract: | Objective To construct microRNA(miRNA) eukaryotic expression vector via pcDNATM 6.2-GW/EmGFP miR eukaryotic expression vector aimed at human Nrf2 gene and to primarily select effective miRNA expression vector for in vitro experiment.Methods Computer-designed oligo nucleotide sequences expressing the pre-miRNA were cloned into plasmid pcDNA6.2-GW/Em-GFP-miR with T4 DNA ligase after annealing.Then enzyme cutting method and sequencing were performed to evaluate the four recombinants named pcDNA-Nrf2-A,pcDNA-Nr... |
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| Keywords: | Nrf2 microRNA eukaryotic expression vector MCF-7 cell |
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