| siRNA靶向慢病毒对人喉癌Hep-2细胞系FAK基因的沉默效应 |
| 投稿时间：2010-07-29 修订日期：2010-09-25 点此下载全文 |
| 引用本文：徐炜,顾栋桦,陈英武,平金良,姚根有.siRNA靶向慢病毒对人喉癌Hep-2细胞系FAK基因的沉默效应[J].医学研究杂志,2011,40(3):85-90 |
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| 基金项目:湖州市科技计划资助项目（2008GS07） |
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| 中文摘要:目的探讨siRNA靶向慢病毒对人喉癌Hep-2细胞系FAK基因的沉默效应，以期探索喉鳞癌基因治疗的新途径。方法Western Blot检测人喉鳞状细胞癌细胞系Hep-2中的FAK蛋白表达水平。用FAK-RNAi慢病毒转染Hep-2细胞，同时监测转染效率。半定量RT-PCR检测FAK基因被FAK-siRNA沉默后mRNA的表达水平。Western-blotting检测FAK基因被FAK-siRNA沉默后蛋白质的表达水平。采用荧光PI染色检测凋亡细胞数目，MTT法检测细胞体外增生能力。结果Western blot结果显示Hep-2细胞中FAK的3个蛋白片段表达量较高。多次重复western blot检测转染前后Hep-2细胞的FAK基因蛋白表达发现，FAK-RNAi慢病毒对目的基因在Hep2细胞中具有显著knockdown作用。使用RT-PCR技术检测FAK-siRNA干扰Hep-2细胞系前后FAK mRNA的表达发现，FAK-RNAi慢病毒对目的基因，在mRNA水平有明显knockdown作用。PI染料染色后，可见FAK-RNAi组30%~35%的肿瘤细胞凋亡。MTT Assay 结果显示，FAK-siRNA慢病毒转染Hep2细胞后，Hep2细胞活力明显下降。结论经用慢病毒载体介导的FAK-siRNA在喉鳞癌细胞中可获得高效转染，并能产生特异性的基因沉默效应。 |
| 中文关键词:喉肿瘤 RNA干扰 黏着斑激酶 基因沉默 |
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| Silencing Effect of siRNA on FAK Geng of Human Laryngeal Carcinoma Hep-2 Cell Line |
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| Abstract:ObjectiveTo investigate the inhibitory effect of gene silencing mediated by FAK-siRNA for laryngeal carcinoma hep-2 cell line, and try to look for a new approach of gene therapy of laryngeal squamous cell carcinoma (LSCC) by synthesizing and filtrating effective FAK-siRNA in vitro. MethodsThe level of FAK protein expression in Hep-2 cells was analyzed with the Western Blot technique. FAK-siRNA lentivirus were transfected into Hep-2 cells. Efficiency of transfection was detected by counting cells. Semi-quantitative analysis of the level of FAK-mRNA expression was performed using RT-PCR. The level of FAK protein expression was analyzed with Western Blot. Cell proliferation viability was tested by MTT assay. ResultsWestern blotting results showed overexpression of three protein fragments of FAK in the Hep-2 cells. The content of protein and mRNA of FAK in the FAK-siRNA lentivirus transfected Hep-2 cells was obviously reduced compared with blank control cells. PI staining in the FAK-siRNA lentivirus transfected cell showed 30%~35% of apoptotic cells in Hep-2 cells.Comparing with the parental Hep2 cells, the transfected cells exhibited a slower growth rate. ConclusionEligible FAK-siRNAs synthesized in vitro mediated with lentiviral vector can be highly effectively transfected into LSCC cells, and induce post-transcriptional gene silencing. |
| keywords:Laryngeal neoplasm RNA interference Focal adhesion kinase, FAK Gene silenceRNAi |
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