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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si RNA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si RNA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Western blot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5.31×10~8TU/m L,1.49×10~9TU/m L及8.50×10~8TU/m L,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si RNA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.
Objective: Hegdehog signaling pathway plays an important role in the development of pancreatic cancer, and its regulation mechanism will lay a good theoretical foundation for elucidating the pathogenesis of pancreatic cancer.To construct a lentiviral vector carrying SMO targeting si RNA, And confirmed its inhibitory effect on the expression of SMO gene in pancreatic cancer cells.Methods: Three SMO gene-targeted si RNA fragments were designed and synthesized, and the lentiviral vector carrying these three fragments was constructed by gene recombination technology and its titer was determined . The constructed lentiviral expression vector was transfected into human pancreatic cancer cell line SW1990. The expression of SMO gene was detected by RT-PCR to screen out the most effective interference expression vector. After transfecting SW1990 cells with this vector, blot was used to detect the expression of SMO protein.Results: The results of gene sequencing and restriction endonuclease digestion suggested that SMO targeted siRNA fragment was successfully synthesized and correctly inserted into the lentiviral expression vector, with no base deletion missense mutation and mutation. The virus titer was 5.31 × The inhibitory rates of SMO gene expression in SW1990 cells were 86.00%, 74.85% and 19.22%, respectively. The inhibitory rates of SMO gene expression were 10 ~ 8TU / m L, 1.49 × 10 ~ 9TU / m L and 8.50 × 10 ~ 8TU / ,effect The inhibition rate of SMO protein expression in SW1990 cells transfected with the optimized expression vector was> 80% .Conclusion: The lentiviral vector carrying SMO gene-targeted si RNA was successfully constructed and the vector could effectively inhibit the expression of SMO Gene expression, for further research provides a good experimental tool.