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目的构建沉默信息调节因子(silent information regulaor,Sir)样蛋白1(Sirtuin 1,SIRT1)的特异性短发夹RNA(shRNA)慢病毒载体,获得敲低SIRT1的肝癌细胞系,以探讨其对肝癌细胞的增殖和耐药敏感性的作用。方法设计针对SIRT1靶点特异性的干涉序列,连接到经HpaⅠ和XhoⅠ双酶切的pSicoR-GFP载体,慢病毒包装293T产生病毒,感染肝癌细胞,建立肝癌细胞SIRT1低表达的稳定株。利用实时定量PCR检测SIRT1的干涉效果;通过平板克隆形成实验、CCK8细胞增殖实验检测SIRT1被干涉后对肝癌细胞增殖能力的影响;通过细胞耐药敏感性实验及实时定量PCR检测耐药基因的表达,考察SIRT1干涉对肝癌细胞耐药敏感性的影响。结果实时定量PCR实验证明该慢病毒干涉载体能显著抑制肝癌细胞中SIRT1的表达,SIRT1干涉可抑制肝癌细胞的增殖能力,下调耐药基因的表达,增强肝癌细胞的药物敏感性。结论 SIRT1抑制肝癌细胞耐药性。
Objective To construct specific short hairpin RNA (shRNA) lentiviral vector of silent information regulaor (Sir) -like protein 1 (SIRT1) and obtain the SIRT1-knockdown hepatocellular carcinoma cell line to investigate its effect on hepatocellular carcinoma The role of cell proliferation and drug sensitivity. METHODS: The target sequence specific to SIRT1 was designed and ligated into pSicoR-GFP vector digested with HpaI and XhoI. 293T virus was packaged in lentivirus to infect hepatoma cells and a stable SIRT1 low expression strain was established. Real-time quantitative PCR was used to detect the interference effect of SIRT1. The effects of SIRT1 on the proliferation of hepatocellular carcinoma cells were detected by plate clone formation assay and CCK8 cell proliferation assay. The expression of drug resistance genes was detected by cell sensitivity test and real-time quantitative PCR , Investigate SIRT1 interference on the sensitivity of hepatocellular carcinoma cells. Results Real-time PCR showed that the lentiviral vector significantly inhibited the expression of SIRT1 in hepatoma cells. SIRT1 interference inhibited the proliferation of hepatoma cells, down-regulated the expression of drug-resistant genes and enhanced the drug sensitivity of hepatocellular carcinoma cells. Conclusion SIRT1 can inhibit the drug resistance of hepatoma cells.