| BackgroundColorectal cancer is one of the most familiar malignant cancers. According to recent data, colorectal cancer is the second tumor death reason next to lung cancer in Europe and Amercia country, is 4th to 6th in our country, and the incidence still increases year after year. Though many therapeutic methods have been made progress to treat colorectal cancer in past 20 years, no one shows a notable improvement to the whole survival rate. So, it is critical to find out a therapeutic method with maximal efficiency and mild toxicity.It is know that apoptosis plays an important role in tumorigenesis and inducing apoptosis of malignant cells. Gene therapy, by introducing proapoptotic genes, attract people's interests more and more.TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand), is a signal molecule which shares many characteristics of tumor necrosis factor(TNF) family members and appears to induce apoptosis only in tumorigenic or transformed cells but not in most of normal cells. It has known that TRAIL was expressed in the majority of human tissue and induce apoptotic cell death by interacting with its receptors.We and other investigators show that TRAIL gene transfered to tumorigenic cells from adenovirus vector can induce apoptosis and cause regression of tumor. We also demonstrate that human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the apoptotic gene to cancers. Here, we use TRAIL gene expressed from the hTERT promoter to assess its anti-colorectal cancer cell activity and the toxicity to the normal human cells.Materials and methodsMaterial Cell lines: 293 cell, Human colorectal cancer line DLD1, HT-29, Normal human bone marrow fibroblast(BMFB) and Normal human smooth muscle cell(NSMC); Adenoviral vectors: Ad/hTERT-gTRAIL, Ad/GT-TRAIL, Ad/PGK-GV16, Ad/CMV-GFP; And other experiment apparatus, materials for cell culture.Methods l)The expansion, purification, titration and qualityanalysis of all the vectors: The vectors, include Ad/hTERT-gTRAIL, Ad/GT-TRAIL, Ad/PGK-GV16 and Ad/CMV-GFP, were all expanded in 293 cells, purified by CsCl banding, titrated by mensurating the OD value of A260. 2) Transgene expression of TRAIL: 3 105 DLD-1, HT29, BMFB, NMSC were inoculated and infected with Ad/hTERT-gTRAIL,Ad/GT-TRAIL+Ad/PGK-GV16 (negative control), Ad/CMV-GFP (vector control) at MOI of 1000 and PBS (blank control), cultured for 48hrs, gene expression was checked by fluorescence microscope and determined by fluorescence-activated cell sorting (FACS) analysis, 3) Cell viability: 5 X 103 DLD-K HT29> BMFB, NMSC were inoculated in 96-well plate, 4 parallel team were set up, 24hrs after inoculation, cells were infected with Ad/hTERT-gTRAIL, Ad/GT-TRAIL+Ad/PGK-GV16 (positive control), Ad/CMV-GFP (vector control) at MOI of 1000 and PBS (blank control), at 24hr, 48hr and 96hr after infection, the cell viability was determined by MTT assay. 4 ) Apoptosis inducing effect: 3 X 105 DLD-1 , HT29, BMFB, NMSC were inoculated in 6-well plate, 24hrs after inoculation, cells were infected with the adenoviruses Ad/hTERT-gTRAIL , Ad/GT-TRAIL+Ad/PGK-GV16 (positive control), Ad/CMV-GFP (vector control) at MOI of 1000 and PBS (blank control) . After infected, the cell morphologies were checked with inverted microscope every day, and 48hrs late, cells were collected and labeled with propidium iodide (PI), subjected to fluorescence-activated cell sorting (FACS) analysis. 5) Statistics analysis: SPSS 10.0 software was used in the stastistic analysis , the cell viability and the cell apoptosis precentage were determined by paired T-test, Statistical significance was claimed when p<0.05.Results.1. Results of transgene expression of TRAIL : GFP/TRAIL expression was observed under fluorescence microscope in DLD-K HT29 but not inBMFB, NMSC. The express percentage of DLD-1, HT29, BMFB, NMSC were 41.6% , 31.4%, 2.77% and 4.64% by FACS analysis. The result show that TRAIL gene with hTERT promoter ca...
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