MEK2基因siRNA的构建和筛选及其在人胶质瘤细胞中的表达  

Screening and construction of MEK2-siRNA and its expression in human glioma

作　　者：贺华[1,2] 陶帮宝[1] 胡国汉[1] 骆纯[1] 李兵[1] 王君玉[1] 卢亦成[1] 

机构地区：[1]第二军医大学长征医院神经外科,上海神经外科研究所,上海200003 [2]干细胞与医学研究生创新实验中心

出　　处：《中华神经医学杂志》2011年第4期356-359,共4页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金，第二军医大学干细胞与医学研究中心研究生创新实验课题

摘　　要：目的构建MEK2．siRNA表达质粒，转染胶质瘤U87细胞并研究其对内源性MEK2表达的敲减效果。方法化学合成4条60个碱基并能转录siRNA发卡结构的DNA寡核苷酸，退火形成2条双链DNA．以T4连接酶接人BgtⅡ和HindⅢ双酶切后的pSUPER．basic载体中。EcoRⅠ和HindⅢ双酶切鉴定出阳性重组克隆，抽提质粒并转染胶质瘤U87细胞，Western blotting检测MEK2蛋白的表达水平。结果酶切鉴定和测序结果表明MEK2-siRNA质粒构建成功，Western blotting结果筛选出较好的MEK2-siRNA质粒。未转染阴性对照质粒MEK2表达水平为0．105±0．023，转染筛选质粒后表达水平为0．030±0．006。结论成功构建了MEK2-siRNA真核表达载体，并证明其能下调胶质瘤U87细胞中MEK2蛋白的表达水平，为RNA干扰技术应用于胶质瘤的基因治疗提供了一定的实验依据．Objective To construct a eukaryotic expression vector for MEK2-siRNA to explore the expression of this endogenous MEK2 in glioma U87 cell line. Methods Four single-stranded template DNAs encoding siRNA against MEK2, each consisting of 60 bp, were synthesized chemically; based on these 4 single-stranded template DNAs, 2 double-stranded DNAs were formed by annealing, then identified by restriction analysis and inserted into vector pSUPER.basic by T4 ligase. Positive recombinants were indentified by EcoRI and HindIII double digestion and transfected into the U87 cells. The protein expression level of MEK2 was determined by using Western blotting. Results Both restriction analysis and sequencing proved that the eukaryotic expression vector for MEK2-siRNA was constructed correctly. MEK2-siRNA plasmid screened out by Western blotting. The MEK2 expression level in negative control group was （0.105±0.023） and that in transfected group was （0.030±0.006）. Conclusion The eukaryotic expression vector for MEK2-siRNA is successfully constructed, which down-regulates the transcription of MEK2 protein in U87 cells. It provides a certain experimental basis for gene therapy of glioma by RNAi technique.

关 键 词：RNA 小分子干扰 神经胶质瘤 MEK2 真核表达载体 

分 类 号：R739.41[医药卫生—肿瘤]