蛋白酶激活受体-2基因靶向shRNA表达质粒的构建及转染食管癌细胞致沉默效应的研究  

Construction of PAR-2 targeted shRNA expression plasmids and study on its silence effect by transfecting esophageal cancer cells

作　　者：陈金梅[1] 邓全军[1] 谢立群[1] 赵建业[1] 刘彩虹[1] 郑艳敏[1] 

机构地区：[1]武警医学院附属医院消化内科,天津市300162

出　　处：《实用医学杂志》2013年第3期349-351,共3页The Journal of Practical Medicine

基　　金：武警医学院附属医院种子基金项目(编号:FYM201115)

摘　　要：目的:构建靶向蛋白酶激活受体-2(PAR-2)基因的shRNA表达质粒,建立PAR-2基因沉默的食管癌EC109稳定细胞株。方法:针对人PAR-2的mRNA序列,设计并体外合成编码shRNA的两条特异性寡核苷酸序列,分别插入pGFP-V-RS质粒中构建2个shRNA表达质粒(PAR-2shRNA-1、PAR-2shRNA-2),经过鉴定,然后将表达质粒转染至食管癌细胞EC109中,经嘌呤霉素筛选,获得稳定干扰的细胞株,运用RT-PCR和WesternBlot检测PAR-2的表达。结果:PAR-2shRNA-1转染人食管癌细胞EC109后,PAR-2基因表达在mRNA和蛋白质水平都受到显著抑制,差异有统计学意义(P<0.05)。结论:成功构建针对PAR-2基因的shRNA表达质粒,转染食管癌细胞EC109,能有效抑制该细胞株PAR-2基因的表达。Objective To construct the PAR-2 targeted shRNA expression plasmids, and to construct the esophageal cancer cells lines with PAR-2 gene silence expression. Methods According to the human PAR-2 mRNA, two oligonucleotide sequences coding shRNA were designed and synthesized in vitro were inserted respectively into pGFP-V-RS to construct two shRNA expression plasmids （PAR-2 shRNA-1, PAR-2 shRNA-2）. After identification, expression plasmids were transfected into EC109 esophageal cancer cells. Stable interference cell lines were obtained by puromycin selection. Expression of PAR-2 was measured by RT-PCR and Western Blot. Results After PAR-2 shRNA-1 being transfected into EC109 esophageal cancer cells, the expression of PAR-2 gene was significantly inhibited at the mRNA and protein level （P 〈 0.05）. Conclusion The PAR-2 shRNA plasmid was constructed successfully. PAR-2 protein expression in EC109 was inhibited by RNAi.

关 键 词：食管肿瘤 EC109 小干扰RNA 蛋白酶激活受体2 基因沉默 

分 类 号：R735.1[医药卫生—肿瘤]