| BackgroundLiver cancer is one of the most malignant cancers with poor prognosis, and about second thirds patients betide in China. Though there have been many therapeutic methods to resist, no one shows notable effect other than operation, but the patients who can be radical operated are less than 15%. It is now considered that apoptosis plays an important role on tumogenesis and at the same time, inducing apoptosis of malignant cells has been a prevenance in tumor therapy. TRAIL (Tumor necrosis factor-related necrosis-inducing ligand), first identified by searching an expressed sequence tag (EST) data base with a conserved sequence contained in many tumor necrosis factor (TNF) family members, appears to induce apoptotic cell death only in tumorigenic or transformed cells and not in most of normal cells. Evidence has shown that repeated intravenous injection of a recombinant, biologically active TRAIL protein induces tumor cell2002apoptosis, suppresses tumor progression, and improves the survival of animals bearing solid tumors without causing any detectable toxicity in nonhuman primates. Therefore, it appears that TRAIL may act as a potent anticancer agent without causing significant toxicity to most normal tissues. Furthermore, TRAIL can elicit an apoptotic bystander effects on malignant cells. The research of anti-liver cancer activity and bystander effect of TRAIL is not familiar in the world. Here, we use binary adenoviral vector system to express human TRAIL gene, then to assess the anti-liver cancer activity of TRAIL and explore its bystander effects. Furthermore, we assess the toxicity of TRAIL to the normal human bone marrow fibroblast (BMFB).Materials and methodsMaterial Cell lines: 293 cell, Human liver cancer line SMMC7721, Normal human bone marrow fibroblast(BMFB); Adenoviral vectors: Ad/GT-TRAIL, Ad/GT-Bax, Ad/GT-LacZ, Ad/PGK-GV16; And others such as experiment apparatus, materials for cell culture and RT-PCR, and so on.Methods l)The expansion, purification, titration and qualityanalysis of all of the vectors: The vectors, include Ad/GT-TRAIL , Ad/GT-Bax, Ad/GT-LacZ and Ad/PGK-GV16, are all expanded 293 cells, purified by CsCl banding, titrated by mensurating the OD value of A260 and conversion, quality analyzed by being found free of E1+ adenovirus and endotoxin. 2) transgene expression of TRAIL: 1 X 106 SMMC7721 infected with Ad/GT-TRAIL+ Ad/PGK-GV16 or Ad/GT-LacZ +72002Ad/PGK-GV16, cultured for 2 days, then obtain the RNA of cells by TRIZOL method, retro-transcripted to cDNA by RT-PCR, and the cDNA expansion PCR conditions were as followed: 35 cycles,45 s at 95 癈, 45 s at 58癈, 45 s at 72 癈, forward primer 5'-AGA CCT GCG TGC TGA TCG TG-3', and reverse primer 5'-TTA TTT TGC GGC CCA GAG CC-3'. PCR products were separated in 1% agarose gels and visualized by ethidium bromide. 3) Cell viability: 5X103 SMMC7721 and BMFB inoculated in 96-well plate, set up 3 parallel team; The day after inoculation, cells were infected with Ad/GT-TRAIL+Ad/PGK-GV16, Ad/GT-Bax+ Ad/PGK-GV16(positive control) ,Ad/GT-LacZ + Ad/PGK-GV16 (vector control) , and PBS (blank control) at MOI of 500; at the day 0,1,3 and 5 after infection, determined the cell viability by MTT assay. 4 ) Apoptosis: 5 X 104SMMC7721 inoculated in 6-well plate, The day after inoculation, cells were infected with the adenoviruses at MOI of 500. After infected, we observed the cell morphology with inverted microscope every day, and then, at the 5th day cells were labeled with ANNEXIN V and propidium iodide (PI), and subjected to fluorescence-activated cell sorting (FACS) to determine the extent of cell death. 5) Bystander effect: mixed the SMMC7721 /TRAIL cells and SMMC7721 cells with different ratios, SMMC7721 /TRAIL cells occupied 0, 5%, 25%, 50%, 75%, 100%, 1 X 104 mixed cells inoculated in 96-well plate with 3 parallel team each ratio, 4 days later determined the cell viability by MTT assay. 5) Mechanism of bystander effect: we took the old media leached the cell component of t...
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