新型自杀基因linamarase-linamarin系统对人肝癌细胞HepG2体外杀.doc

新型自杀基因linamarase/linamarin系统对人肝癌细胞HepG2体外杀 　 作者：陈明浩, 马俊, 李海民*, 李军, 李韧, 张福琴, 窦科锋 【摘要】 　　目的: 研究新型自杀基因亚麻苦甙水解酶/亚麻苦甙（lis/lin）统对人肝癌细胞HepG2的体外杀伤效应。方法: 应用分子克隆技术构建热带植物木薯的cDNA文库, 并从中扩增出lin基因克隆至pEGFPN1载体, 构建质粒pEGFPN1lis。通过电穿孔法将其转染人肝癌细胞株HepG2, 同时进行G418筛选, 直至出现抗性克隆, 扩大培养, 命名为HepG2/lis, 通过荧光显微镜观察、 RTPCR、 Western blot鉴定lis基因的表达; 采用MTT法观察不同浓度lin对已转染的HepG2的杀伤作用及旁观者效应。结果: RTPCR和Western blot证明转染的lis基因能够在HepG2细胞中表达并产生融合蛋白lisEGFP; 低浓度lin对转染了lis基因的HepG2细胞具有明显的细胞毒效应; 旁观者效应显示仅10%左右的HepG2/lis细胞与90%的HepG2细胞混合培养于lin浓度为500 mg/L的培养基时, 就可显示出明显的旁观者效应。结论: lis/lin自杀基因系统在lin浓度为500 mg/L时对人原发性肝癌细胞HepG2具有明显的杀伤作用和旁观者效应。 【关键词】 人肝癌细胞 HepG2 自杀基因 旁观者效应 　　[Abstract] AIM: To investigate the killing effect of linamarase/linamarin (lis/lin) system on hepatocellular carcinoma cell line HepG2 in vitro. METHODS: A cDNA library was built from RNA of cassava by RTPCR, then the linamarase gene was amplified from it by PCR and cloned into the eukaryotic expression vector plasmid pEGFPN1, which made up the recombinant plasmid pEGFPN1lis. The human HCC cells HepG2 were transfected with the recombinant plasmid mediated by electroporation and screened by G418 to yield the positive clone which was termed HepG2/lis. The expression of lis was confirmed by fluorescent staining, RTPCR and Western blot. The killing effect and bystander effect of linamarin with different concentrations on HepG2 was detected by MTT. RESULTS: RTPCR confirmed the expression of lis gene in HepG2 and Western blot analysis confirmed existence of lisEGFP fusion protein in HepG2. Linamarin in low concentration had shown notable cytotoxic effect on HepG2/lis. When HepG2/lis cells were mixed with parental HepG2 cells at a ratio of 10∶90 and cultivated in 500 mg/L lin medium, significant bystander effect was observed in vitro. CONCLUSION: The linamarase/linamarin suicide gene system has strong killing effect and bystander effect on HCCs with the concentration of 500 mg/L lin. 　　[Keywords]hepatocellular carcinoma cell; HepG2; suici