PDF《鉴定和表达》

・ 基础研究 ・ 人单磷酸腺苷激活的蛋白激酶催化亚单位α2基因shRNA载体的 鉴定和表达 卢俊, 徐世元, 崔睿, 张庆国, 雷洪伊 南方医科大学珠江医院麻醉科, 广东 广州

510282 摘要: 目的 构建针对人单磷酸腺苷激活的蛋白激酶催化亚单位α2 (AMPKα2)基因的pGPU6/GFP/Neo-shRNA表达载体, 转染SH-SY5Y细胞株, 观测其对AMPKα2基因的沉默效果.

方法 利用Ambion在线设计软件设计针对人AMPKα2基因 的shRNA干扰序列, 克隆到pGPU6/GFP/Neo质粒载体上.对重组质粒进行DNA序列测定和酶切分析.用质脂体将重 组质粒转导SH-SY5Y细胞株, 经G418筛选阳性克隆后用RT-PCR和Western blot法检测AMPKα2的mRNA和蛋白表达 水平.结果 经DNA 测序和酶切鉴定表明,

4 个shRNA 表达载体构建成功.4 个重组质粒之一的 pGPU6/GFP/ Neo-shRNA AMPKα2(3)能有效抑制AMPKα2基因在SH-SY5Y细胞株中的表达, 抑制率为63%.结论 成功构建了针对 人AMPKα2基因的shRNA表达载体pGPU6/GFP/Neo-shRNAAMPKα2.pGPU6/GFP/Neo-shRNAAMPKα2(3)能有效抑 制AMPKα2基因在SH- SY5Y细胞株中的表达, 为将来应用其研究AMPK在细胞损伤中的作用奠定了基础. 关键词: 局麻药;

RNAi干扰;

单磷酸腺苷激活的蛋白激酶催化亚单位α2 中图分类号: Q789 文献标志码: A 文章编号: 1673-4254 (2011) 01-0086-04 Identification and expression of shRNA vectors targeting human AMPKα2 LU Jun, XU Shi-yuan, CUI Rui, ZHANG Qing-guo, LEI Hong-yi Department of Anesthesiology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China Abstract: Objective To construct pGPU6/GFP/Neo-shRNA expression vector targeting human AMPKα2 gene and evaluate its silencing effect in SH-SY5Y cell line. Methods The oligonucleotides designed by Ambion online CAD software targeting AMPKα2 were cloned into the pGPU6/GFP/Neo vector. After confirmation by DNA sequencing and enzyme digestion analysis, the recombinant vectors were transfected into the SH-SY5Y cell line via lipofectamine and the positive clones were selected using G418. The expression levels of AMPKα2 mRNA and protein in the transfected cells were detected by RT-PCR and Western blotting, respectively. Results Four shRNA vectors were successfully constructed as confirmed by DNA sequencing and the enzyme digestion analysis. Among the

4 recombinant vectors, pGPU6/GFP/Neo-shRNA AMPKα2(3) showed the strongest gene silencing effect and down-regulated the protein expression of AMPKα2 by 63% in the transfected cells. Conclusion Transfection with pGPU6/GFP/Neo-shRNA AMPKα2(3) results in effective inhibition of AMPKα2 gene expression in SH-SY5Y cells, which provide a means for studying AMPK-mediated cell injury. Key words: local anesthetic;

RNA interference;

AMPK 单磷酸腺苷激活的蛋白激酶(AMPK)是一种丝/ 苏氨酸蛋白激酶, 参与各种生理及病理过程中细胞 能量代谢的调控.在正常生理条件下, AMPK 处于 无活性状态, 当细胞内ATP不足时, AMP/ATP比率增 高, 激活 AMPK 激酶系统, 一方面抑制合成代谢途 径, 以减少 ATP 的消耗, 另一方面促进分解代谢途 径.因此, AMPK 被称为 能量代谢总开关 [1-2] . 研 究发现, 局麻药可通过干扰氧化磷酸化和抑制线粒 体呼吸链复合物I, 使ATP生成减少, 并引起细胞内活 性氧族(ROS)明显增多, 从而损伤细胞[3-5] .有研究表 明, AMPK 的持续激活可引起 ROS 产生增加[6] .因此, AMPK与局麻药引起的细胞损伤密切相关. 本研究通过构建4个针对AMPKα2 基因的shRNA 真核表达载体并检测其对 SH- SY5Y 细胞株 中AMPKα2 基因的沉默作用, 为进一步研究 AMPK 在局麻药所致细胞损伤中的作用奠定了基础.