survivin shRNA表达质粒对GBC-SD细胞生长的抑制及对化疗敏感性的影响  

The effect of survivinshRNA on chemotherapy resistance of GBC-SD cells

作　　者：胡松 蔡晓棠[2] 沈汉斌 张少炎[2] 楼朝阳[2] 

机构地区：[1]湖北省武汉市第五医院普外I科,湖北武汉430050 [2]解放军第四七七医院普通外科,湖北襄樊441003

出　　处：《中国普通外科杂志》2011年第2期154-158,共5页China Journal of General Surgery

摘　　要：目的构建针对survivin基因的shRNA真核表达载体,观察重组质粒pEGFP-survivin对胆囊癌细胞(GBC-SD)化疗敏感性的影响。方法设计、合成包含BbsI酶切位点的针对survivin的shRNA,与BbsI酶切后真核表达载体pEGFP-H1连接,将其定向克隆至H1启动子下,构建成重组载体pEGFP-survivin;采用脂质体法将pEGFP-H1和重组质粒导入GBC-SD细胞中;用G418对转染的细胞进行稳定筛选。用RT-PCR测各组细胞中survivinmRNA的表达;以合适浓度的顺铂(DDP)(3.0μg/mL)作用相同时间后,用MTT法检测GBC-SD,GBC-SD/EGFP,GBC-SD/survivin 3种细胞存活率,TUNEL法观察细胞调亡。结果 pEGFP-survivin成功构建。GBC-SD/survivin细胞中的survivin表达水平较其余2种细胞明显下降(分别下降74.7%和71.5%);经DDP作用后,GBC-SD/survivin细胞存活率较其他2组明显降低,3种细胞均可见棕色凋亡细胞核。结论成功构建了针对survivin基因的shRNA真核表达载体,并获得稳定表达survivin shRNA的细胞株GBC-SD/survivin。survivin shRNA能明显降低GBC-SD细胞中survivin的表达,提高其对化疗药物的敏感性。Objective To construct survivinshRNA expression vector and investingate the effect of survivinshRNA on chemotherapy resistance of GBC-SD cells.Methods The siRNA sequence targeting survivinmRNA was synthesized and cloned into pEGFP-H1.The constructed plasmid and pEGFP-H1 were transfected into GBC-SD cells respectively via liposome.Then the transfected cells were selected with G418.GBC-SD cells were divided into three groups： GBC-SD,GBC-SD/EGFP and GBC-SD/survivin groups.SurvivinmRNA was tested by RT-PCR.Then cells of the 3 groups were treated with adequate concentration of DDP（3.0 μg/mL） for similar periods of time,cell survival rate was detected with MTT and apoptosis was observed by TUNEL.Results The recombinant plasmid,pEGFP-survivin,was successfully constructed.Compared to the other 2 groups,the level of survivin expression in GBC-SD/survivin group was obviously decreased（74.7%,71.5%）.After DDP treatment,cell survival rate was obviously decreased in GBC-SD/survivin group compared with other 2 groups.There were brownly apopotosis nucleuses in the three groups.Conclusions SurvivinshRNA expression vector has been constructed successfully and GBC-SD cells with stable expressionshRNA has been obtained.The survivinshRNA could significantly down-regulate the expression of survivin in GBC-SD cells and improve the sensibility to chemotherapy.

关 键 词：胆囊肿瘤 RNA干扰 SURVIVIN 短发夹环RNA 胆囊癌细胞 

分 类 号：R735.8[医药卫生—肿瘤]