慢病毒载体介导RNAi靶向抑制胰腺癌细胞survivin表达并诱导细胞凋亡的实验研究
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李宜雄E-mail:liyixiong6@hotmail.com

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国家自然科学基金资助项目(30872492);湖南省自然科学基金资助项目(08JJ3042)。


Lentiviral vector-mediad RNA interference targeted against survivin inhibits survivin expression and induces cell apoptosis of human pancreatic cancer in vitro
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    摘要:

    目的:探讨运用慢病毒载体介导的RNA干扰技术对survivin的抑制效率及对胰腺癌细胞SW1990凋亡的影响。
    方法:应用pGCSIL-neo质粒构建3对针对survivin的慢病毒ShRNA载体,转染包装细胞293T,收集病毒上清转染胰腺癌细胞系SW1990,实时荧光定量PCR和western-blot免疫印迹检测癌细胞内survivin的表达; 测定caspase3/7活性和DAPI染色检测细胞凋亡。
    结果:成功构建3个survivin-ShRNA慢病毒载体,LV-shRNA-survivin-1对survivn的mRNA和蛋白表达抑制效率分别为73.50%和87.64%;与对照组相比,其caspase3/7活性升高14.5倍,细胞凋亡明显增加(11.95%)。
    结论:慢病毒载体介导的Suvivin-ShRNA靶向转染可有效抑制survivin表达,并显著增加胰腺癌细胞的凋亡。

    Abstract:

    Objective:To investigate the possibility of survivin inhibition by lentiviral vector-mediated RNA interference and the influence on cell apoptosis in pancreatic cancer cell line.
    Methods:The lentiviral vector of SiRNA targeted against survivin(LV-shRNA-survivin-1, LV-shRNA-survivin-2, LV-shRNA-survivin-3) was constructed and transfected into the packaging cells 293T, and then the supernatant with virus was collected to transfect SW1990 cells. Quantitative real-time fluorescent PCR and Western-blot were used to detect the expression of survivin. DAPI staining and detection of enzymatic activity of caspase 3/7 were employed to examine cell apoptosis.
    Results:Three lentiviral vector-survivin-shRNA were constructed successfully. In the LV-shRNA-survivin-1 group,the survivin mRNA and protein expression inhibitory rate was 73.50% and 87.64% respectively; when compared to control group, the activity of caspase-3/7 increased significantly,which showed a 14.5-fold increase, and apoptosis increased 11.95%.
    Conclusions:Lentiviral vector-mediad RNA interference targeted against survivin can effectively inhibit survivin expression and increase cell apoptosis significantly.

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易小平|江春|宰红艳|邓公平|李宜雄.慢病毒载体介导RNAi靶向抑制胰腺癌细胞survivin表达并诱导细胞凋亡的实验研究[J].中国普通外科杂志,2010,19(3):227-233.
DOI:10.7659/j. issn.1005-6947.2010.03.001

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  • 收稿日期:2009-12-04
  • 最后修改日期:2010-02-17
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  • 在线发布日期: 2010-03-15