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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pc DNA3.0-Alu(p Alu)重组质粒、右臂缺失突变体pc DNA3.0-Alu-left arm(p Alu-LA)、G25C突变体pc DNA3.0-Alu G25C(p Alu-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p Alu、p Alu-LA、p Alu-M经酶切及DNA测序鉴定符合预期;p Alu、p Alu-LA转染显著促进A549细胞ROS产生;与p Alu相比,p Alu-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.“,”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 H2 O2 treated A549 cells. pc DNA3. 0-Alu (p Alu), pc DNA3. 0-Alu-left arm (p Alu-LA) and pc DNA3. 0-Alu G25 C (p Alu-M) were constructed by directional cloning and point mutation. After recombinant transfection, reactive oxygen species (ROS) produced in transfected A549 cells was detected by DCFHDA. Results:Alu fragment was successfully amplified from stressed A549 cells. Restriction analysis and DNA sequencing proved that recombinant eukaryotic expression plasmids of p Alu, p Alu-LA and p Alu-M were constructed as expected. p Alu or p Alu-LA transfection promoted ROS production significantly in A549 cells, while p Alu-M transfection induced ROS production was markedly decreased as compared with p Alu 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.