中国普通外科杂志 ›› 2011, Vol. 20 ›› Issue (2): 154-158.

• 基础研究 • 上一篇    下一篇

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

胡松1|蔡晓棠2|沈汉斌1|张少炎2|楼朝阳2   

  1. (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)
  • 收稿日期:2010-04-26 修回日期:2010-10-12 出版日期:2011-02-15 发布日期:2011-02-15
  • 通讯作者: 沈汉斌 E-mail:shbsqj@yahoo.com.cn
  • 作者简介:胡松|湖北省武汉市第五医院副主任医师|主要从事普外胃肠方面的研究。

The effect of survivin shRNA on chemotherapy resistance of GBC-SD cells

HU Song1, CAI Xiaotang2, SHEN Hanbin1, ZHANG Shaoyan2, LOU Chaoyang2   

  1. (1.Department of General Surgery, the Fifth Hospital of Wuhan, Wuhan 430050, China; 2.Department of General Surgery, the 477th Hospital of People’s Liberation Army,Xiangfan|Hubei 441003, China)
  • Received:2010-04-26 Revised:2010-10-12 Online:2011-02-15 Published:2011-02-15

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survivin mRNA的表达;以合适浓度的顺铂(DDP)(3.0 μg/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。

关键词: 胆囊肿瘤; RNA干扰; survivin; 短发夹环RNA; , 胆囊癌细胞

Abstract:

Objective:To construct survivin shRNA expression vector and investingate the effect of survivin shRNA on chemotherapy resistance of GBC-SD cells.
Methods:The siRNA sequence targeting survivin mRNA 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. Survivin mRNA 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:Survivin shRNA expression vector has been constructed successfully and GBC-SD cells with stable expression shRNA has been obtained. The survivin shRNA could significantly down-regulate the expression of survivin in GBC-SD cells and improve the sensibility to chemotherapy.

Key words: Gallbladder Neoplasms, RNA Interference, Survivin, shRNA, GBC-SD Cell

中图分类号: 

  • R 735.8

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