文章摘要
雷恩宇,毛 壮,纪奇峰,束 毅,王 伟,颜 真.Alu RNA及其突变体真核表达质粒构建以及对A549细胞活性氧簇产生的影响[J].南京医科大学学报,2018,(5):577~581
Alu RNA及其突变体真核表达质粒构建以及对A549细胞活性氧簇产生的影响
Construction of eukaryotic expression plasmids for Alu RNA and its mutants and the effect on ROS production in A549 cells
投稿时间:2017-09-23  
DOI:10.7655/NYDXBNS20180502
中文关键词: Alu RNA  氧化应激  A549细胞  基因重组  活性氧簇
英文关键词: Alu RNA  oxidative stress  A549 cell  gene recombination  reactive oxygen species
基金项目:国家自然科学基金(81702733);肿瘤生物学国家重点实验室项目(CBSKL2015Z14,CBSKL201713);陕西省科技统筹创新工程(2016KTCL03-09)
作者单位
雷恩宇 空军军医大学药学院药物基因组学教研室陕西 西安 710032 
毛 壮 空军军医大学药学院药物基因组学教研室陕西 西安 710032 
纪奇峰 空军军医大学药学院药物基因组学教研室陕西 西安 710032 
束 毅 空军军医大学药学院药物基因组学教研室陕西 西安 710032 
王 伟 空军军医大学药学院药物基因组学教研室陕西 西安 710032 
颜 真 空军军医大学药学院药物基因组学教研室陕西 西安 710032 
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中文摘要:
      目的:从氧化应激的A549细胞鉴定Alu RNA表达,构建Alu RNA及其2种功能缺失突变体的真核表达质粒,探讨过表达Alu RNA及其突变体对细胞氧化应激的影响。方法:设计Alu RNA特异性引物,H2O2应激A549细胞,RT?PCR扩增目的片段,通过定向克隆、点突变等方法构建pcDNA3.0?Alu(pAlu)重组质粒、右臂缺失突变体pcDNA3.0?Alu?left arm(pAlu?LA)、G25C突变体pcDNA3.0?Alu G25C(pAlu?M);转染A549细胞,DCFH?DA检测活性氧簇(reactive oxygen species,ROS)产生水平。结果:从应激的A549细胞成功扩增了Alu分子,重组的真核表达质粒pAlu、pAlu?LA、pAlu?M经酶切及DNA测序鉴定符合预期;pAlu、pAlu?LA转染显著促进A549细胞ROS产生;与pAlu相比,pAlu?M转染诱导产生的ROS显著降低。结论:成功克隆了氧化应激刺激下A549细胞产生的Alu RNA及其2种功能缺失突变体的真核表达质粒,体外实验显示Alu RNA通过翻译水平调控促进A549细胞ROS产生。
英文摘要:
      Objective:To identify Alu RNA expressed in oxidative?stressed A549 cells and to construct eukaryotic expression plasmids for Alu RNA and its two kinds of loss?of?function mutants so as to study the effect on oxidative stress in cells with overexpression of Alu RNA and its mutants. Methods:Alu RNA was amplified with RT?PCR from H2O2 treated A549 cells. pcDNA3.0?Alu(pAlu),pcDNA3.0?Alu?left arm(pAlu?LA)and pcDNA3.0?Alu G25C(pAlu?M)were constructed by directional cloning and point mutation. After recombinant transfection,reactive oxygen species(ROS) produced in transfected A549 cells was detected by DCFH?DA. Results:Alu fragment was successfully amplified from stressed A549 cells. Restriction analysis and DNA sequencing proved that recombinant eukaryotic expression plasmids of pAlu,pAlu?LA and pAlu?M were constructed as expected. pAlu or pAlu?LA transfection promoted ROS production significantly in A549 cells,while pAlu?M transfection induced ROS production was markedly decreased as compared with pAlu transfection. Conclusion:The eukaryotic expression plasmids for Alu RNA and its loss?of?function mutants were successfully constructed from oxidative?stressed A549 cells. Data in vitro suggested that Alu RNA promoted ROS production in A549 cells with translational regulation.
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