The Killing Effect And Bystander Effect Of Linamarase/Linamarin Suicide Gene System On Human Hepatocellular Carcinoma In Vitro

Objective:The current studies were designed to make up the hepatoma carcinoma cell lines HepG2/lis and SMMC/lis which can stably express the lis gene, then to investigate the killing effect and bystander effect of linamarase/linamarin ( lis/lin ) system on hepatocellular carcinoma in vitro.Methods:1 The human HCC cells HepG2 and SMMC-7721 were transfected with the recombinant plasmid pEGFP-N1-lis and plasmid pEGFP-N1 mediated by electroporation and screened by G418 to yield the positive clones which were termed HepG2/lis, HepG2/EGFP, SMMC/lis, SMMC/EGFP. Then the expression of lis was confirmed by fluorescent staining and RT-PCR.2 to examine the difference in characteristics of these genetically modified cells by set out growth curve; to observe the cytotoxicity of linamarin with different high concentrationgs on normal HepG2 and SMMC-7721 cells by MTT; the killing effect of linamarin with different low concentrations on hepatocellular carcinoma cells in different time were detected by MTT.3 to detecte the bystander effect of lis/lin suicide gene system by combining the genetically modified cells with normal cells under different proportion and culturing these cells with lin.Results:1 The HepG2/lis and SMMC/lis which can stably express lis gene were gained by electroporation and G418 screening. Fluorescent staining confirmed the existence of lis-EGFP fusion protein in these cells and RT-PCR confirmed the expression of lis gene.2 The growth curve of these cells indicate there is no differences in characteristics of these genetically modified cells. The high concentration of lin has no cytotoxicity on HepG2 and SMMC-7721 which were not modified genetically. But low concentration of lin has significant cytotoxicity on HepG2/lis and SMMC/lis cells. Most cells were killed by lin (100~1000mg/L) in 2 to 4 days.3 When HepG2/lis cells were mixed with parental HepG2 cells at a ratio of 10:90 and cultivated in 500 mg/L lin medium, most cells were killed which meaned significant bystander effect was observed in vitro.Conclusion:1 The linamarase/linamarin suicide gene system has strong killing effect on hepatocellular carcinoma cells in vitro.2 The bystander effect of the inamarase/linamarin suicide gene system is powerful.