组织因子siRNA表达载体的构建及其抑制人神经母细胞瘤细胞组织因子表达的研究

Construction of TFsiRNA Expression Vector and Study on Its Inhibitive Effect in Human Neuroblastoma

目的:构建组织因子(TF)特异性小干扰RNA(siRNA)表达载体,观察其对人神经母细胞瘤细胞TF表达的影响。方法:分子生物学方法构建TFsiRNA-pSUPER表达载体,限制酶切予以初步筛选,基因测序进行证实。以脂质体Lipo-fectamine2000介导转染人神经母细胞瘤细胞系SK-N-MC。以WesternBlotting检测TF表达。结果:所构建TFsiRNA-pSUPER表达载体经基因测序证实。SK-N-MC细胞高表达TF,TFsiRNA-pSUPER表达载体转染48h后,TF表达水平与转染pSUPER质粒的对照细胞相比显著降低,呈剂量依赖性。结论:人神经母细胞瘤SK-N-MC中TF异常高表达,本研究构建的TFsiRNA表达载体可在SK-N-MC中特异性下调TF表达,可作为干预TF表达、研究其非凝血功能的有效手段。

Objective:To construct the specific tissue factor(TF)small interfering(siRNA)expressive vector and study the regulative effect on TF expression in human neuroblastoma cells.Method:TFsiRNA-pSUPER plasmid was construscted by inserting specific 19-nt silencing sequence targeting TF gene into pSUPER vector.Potential construct were screened by restrictive enzyme digestion reaction and then identified by gene sequencing.Transfection of TFsiRNA-pSUPER was performed by using lipofectamine2000.The expression of TF was examined by Western blotting.Results:The constructed TFsiRNA-pSUPER was identified by gene sequencing.Human neuroblastoma cell line SK-N-MC expressed high level of TF.Knockdown of TF expression was achieved by transfection of TFsiRNA-pSUPER on SK-N-MC cells in a dose-deqendent manner.Conclusion:These results suggest that TFsiRNA-pSUPER has been constructed successfully and can knockdown TF in high TF-expressive human neuroblastoma cell line SK-N-MC.TFsiRNA-pSUPER can be used as a potential means of inhibition of TF expression especially while studying its non-coagulative function.