[The killing effect and bystander effect of linamarase/linamarin suicide gene system on human hepatocellular carcinoma cell line HepG2 in vitro].

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.