小干扰RNA抑制胃癌细胞株葡萄糖调节蛋白Grp78的表达  

Repressive effects of small interfering RNA on expression of glucose-regulated protein Grp78 in human gastric cell line

作　　者：张新晨[1] 杨维良[1] 徐华锋[2] 吴德全[1] 杨宝峰[3] 

机构地区：[1]哈尔滨医科大学附属第二医院普通外科,黑龙江省哈尔滨市150086 [2]黑龙江大学生命科学院,黑龙江省哈尔滨市150080 [3]哈尔滨医科大学药理学教研室,150080

出　　处：《世界华人消化杂志》2007年第5期447-452,共6页World Chinese Journal of Digestology

基　　金：黑龙江省自然科学基金资助项目;No.D2004-23~~

摘　　要：目的:观察Grp78靶向RNA干扰质粒载体对人类胃癌细胞株7901 Grp78基因表达的抑制作用.方法:设计1对Grp78基因发卡寡核苷酸,并与psiSTRIKETM质粒载体连接,构建受控于人RNA聚合酶Ⅲ启动子U6的真核表达载体,将重组质粒导入人类胃癌细胞株7901内,分别在转染前、转染后24,48和72h通过RT-PCR、免疫荧光技术检测Grp78mRNA及蛋白水平的表达情况.结果:成功构建RNA干扰质粒载体,靶向Grp78干扰质粒载体命名为psiSTRIKETM/Grp78.将上述质粒转染到人类胃癌细胞株后,观察到Grp78mRNA及蛋白水平的表达明显下调,随着时间的延长Grp78在实验组中的表达逐渐降低,并随着时间推移稳定存在.阴性对照组,转染前组,转染12,48,72h组Grp78的mRNA相对表达量分别为1.069,0.972,0.662,0.408,0.420.转染后组较转染前组及阴性对照组分别具有显著性差异(P<0.01),转染前与阴性对照组无显著性差异(P>0.05).转染前后组免疫荧光表达结果统计学差异明显(P<0.001),其中转染后48h同转染后24h相比较Grp78蛋白表达明显减少(P<0.00714).结论:构建的RNA干扰真核表达载体psiSTRIKETM/Grp78能明显抑制Grp78mRNA及蛋白的表达.AIM： To construct the eukaryotic vector of RNA interference specific expression for glucoseregulated protein Grp78, and to observe silencing effect on Grp78 expression in human gastric cell line 7901. METHODS： Gene sequences of human Grp78 were obtained from GenBank. A couple of Grp78 gene hairpin oligonucleotide was designed, whose sequence met the need of psiSTRIKE^TM U6 small interfering RNA （siRNA） expression vector. The designed oligonucleotide was inserted into plasmid psiSTRIKE^TM, which was an eukaryotic expression vector controlled by the U6 promoter of RNA polymerase Ⅲ, and then human gastric cell line 7901 was transfected with the recombinant plasmid by Lipofectamin2000. The expression of Grp78 was detected both at mRNA and protein level by reverse transcription-polymerase chain reaction （RT-PCR） immunofluorescent assay, respectively, before transfection, 24, 48, and 72 hours after transfection. RESULTS： The recombinant plasmid of RNA interference specific for Grp78 was successfully constructed, named as psiSTRIKE^TM/Grp78. The expression of Grp78 was obviously downregulated both at mRNA and protein level by psiSTRIKE^TM/Grp78 transfection. The analysis for the relative level of Grp78 mRNA showed a gradual and stable decrease with the prolonging of time. The expression of Grp78 mRNA was 1.069, 0.972, 0.662, 0.408, and 0.420, respectively, in the cells of negative control, before transfection, 24, 48, and 72 hours after transfection, and it was significantly lower in the cells after transfection than that before or without transfection （P 〈 0.01）. There was no difference between the negative control group and pre-transfection group （P 〉 0.05）. Immunofluorescent assay showed that the protein expression of Grp78 had been significantly decreased in the cells after transfection than that before transfection （P 〈 0.001）, and it was also markedly different between 48 and 24 hours after transfection （P 〈 0.00714）. CONCLUSION： The siRNA eukaryotic expression vecto

关 键 词：胃癌 RNA干扰 葡萄糖调节蛋白 逆转录-聚合酶链反应 免疫荧光技术 

分 类 号：R735.2[医药卫生—肿瘤]