SIRT1对肝癌细胞耐药敏感性的影响  

Effect of SIRT1 on drug resistance of hepatocellular carcinoma cells

作　　者：纪光红 阎新龙[3] 王海洋[2] 张彪[2] 曾泉[2,4] 周军年[2,4] 范增[2,4] 黄映辉[3] 岳文[2,4] 裴雪涛[2,4] JI Guang-hong YAN Xin-long WANG Hai-yang ZHANG Biao ZENG Quan ZHOU Jun-nian FAN Zeng HUANG Ying-hui YUE Wen PEI Xue-tao(Guangxi Medical University, Nanning 530000, China Laboretory of Stem Cell and Regenerative Medicine, Beijing Institute of Transfusion Medicine, Beijing 100850, China College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China South China Institute of Bio-medicine, Guangzhou 510005, China)

机构地区：[1]广西医科大学,南宁530000 [2]军事医学科学院野战输血研究所干细胞与再生医学研究室,北京100850 [3]北京工业大学生命科学与生物工程学院,北京100124 [4]华南生物医药研究院,广州510005

出　　处：《军事医学》2017年第2期90-95,共6页Military Medical Sciences

基　　金：国家863计划资助项目(2013AA020107);广州市健康医疗协同创新重大专项资助项目(201400000003-1);国家自然科学基金资助项目(81472341);北京市自然科学基金资助项目(7152095)

摘　　要：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 build a short-hairpin RNA（shRNA） interference lenliviral vector of silent information regulator（sir）-like protein 1（Sirtuin 1,SIRT1） gene and to explore its effect on proliferation and drug resistance in hepatocellular carcinoma（HCC） cells.Methods The SIRI1 target specific oligonucleotide sequence was designed and connected to pSicoR-green fluorescent protein（GFP） vector digested by the Hpa I and Xho I.Then,293 T cells were packaged to produce the lentivirus and transfected with liver cancer cells.qRT-PCR assays further indicated that SIRT1 gene was stably knocked down in HCC cells.The effect on cell proliferation was detected by foci formation assay and CCK8 assay.In addition,drug resistance experiments and qRT-PCR were used to detect the effect on resistance of liver cancer cells when the SIRT1 gene was knocked down.Results The result of qRT-PCR demonstrated that the lentivirus shRNA vector of SIRT1 gene was constructed and could suppress the expression level of SIRT1.The proliferation and drug resistance genes of HCC cells were inhibited and drug sensitivity was enhanced.Conclusion SIRT1 gene represses the drug resistance of HCC cells.

关 键 词：沉默信息调节因子样蛋白1 RNA干扰 基因敲除 肝癌 耐药 间充质干细胞 聚合酶链反应 

分 类 号：R735.7[医药卫生—肿瘤] R979.1[医药卫生—临床医学]