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Construction of a recombinant lentiviral vector carrying siRNA targeting SMO gene: Effect on SMO gene expression in pancreatic cancer cells

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Author:: No author available

Journal Title:: World Chinese Journal of Digestology

Issue:: 19

DOI:: 10.11569/wcjd.v24.i19.2974

Key Word:: 胰腺癌;SMO基因;siRNA;慢病毒表达载体;Pancreatic cancer;SMO gene;siRNA;Lentiviral vector

Abstract：目的:Hegdehog信号通路在胰腺癌发生发展过程中发挥着重要的作用,针对其调控机制的研究将对胰腺癌发病机制的阐明奠定良好的理论基础.构建携带SMO靶向siRNA慢病毒表达载体,并证实其对胰腺癌细胞中SMO基因表达的抑制作用.方法:设计并合成3条SMO基因靶向siRNA片段,利用基因重组技术构建携带此3条片段的慢病毒表达载体并测定其滴度,构建好的慢病毒表达载体转染人胰腺癌细胞株SW1990后,采用RT-PCR法检测SMO基因的表达以筛选出效果最佳的干扰表达载体,并以此载体转染SW1990细胞后采用Westernblot法检测SMO蛋白表达.结果:基因测序与酶切电泳结果提示成功合成SMO靶向siRNA片段且其正确插入慢病毒表达载体,无碱基缺失错排与突变,测定病毒滴度分别为5.31×108 TU/mL,1.49×109TU/mL及8.50×108 TU/mL,经转染SW1990细胞后,测定对SMO基因表达的抑制率分别为86,00％、74.85％及19.22％,效果最优的干扰表达载体转染SW1990细胞后对SMO蛋白表达的抑制率＞80％.结论:成功构建携带SMO基因靶向siRNA的慢病毒表达载体,且该载体可有效抑制胰腺癌细胞中SMO基因的表达,为进一步的研究提供了良好的实验工具.

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