【摘要】 目的:针对survivin基因,构建短发卡状RNA干扰(short hairpin RNA interference,shRNA interference)真核质粒表达载体,并转染到高表达survivin基因的舌鳞癌细胞株Tca8113中,筛选出稳定低表达survivin基因的细胞株。方法:针对survivin基因的mRNA序列设计,并合成2对寡核苷酸序列,插入质粒pSilencerTM-2.1U6-neo中,形成重组质粒(PS1和PS2)。用大肠杆菌扩增,酶切、PCR、测序鉴定,用脂质体介导转染Tca8113细胞,G418筛选得到稳定表达株,荧光实时定量PCR和Western印迹检测survivin基因在mRNA和蛋白水平的变化。采用SAS8.0软件包对数据进行t检验。结果:2个真核质粒表达载体均构建成功,并筛选出转染成功的细胞。与对照组相比,PS1和PS2转染的Tca8113细胞中的survivin基因在mRNA水平均显著下降,P<0.05;在蛋白水平,PS2转染后显著下降,P<0.01。结论:本研究成功建立针对survivin基因的shRNA质粒载体,并筛选出稳定低表达survivin基因的Tca8113细胞,为口腔鳞癌的基因治疗提供了理论基础。更多还原

【Abstract】 PURPOSE:To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene,transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level,and choose the cells whose survivin gene were suppressed significantly.METHODS:Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized,and cloned into pSilencerTM-2.1U6-neo plasmid.The recombinant plasmids(named PS1 and PS2) were amplified in Ecoli.DH5α was identified by restriction digestion,PCR and sequencing.The vectors were transfected into Tca8113 cells with lipofectamine 2000.After selection with G418,the stable cell clones were attained.Survivn expression was assayed with real-time quantitative PCR and Western blotting.SAS8.0 software package was used for Student t test.RESULTS:Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained.As compared with those of control,survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed(P<0.05).In protein level,only those of transfected cell with PS2 was significantly suppressed(P<0.01).CONCLUSIONS:The shRNA interference expression plasmid vectors of survivin gene are successfully constructed,and Tca8113 cells which express survivin gene in a stable lower level are attained,which enable us to carry out further research on gene therapy of oral squamous cell carcinoma.更多还原