| Colorectal cancer is both a common cancer and a common cause of cancer death. The world wide incidence of colorectal cancer varies widely between countries and races: high incidence is seen in industrialized Western countries; lower rates are seen in Asia and Africa. The world-wide incidence has increased over the last 30 years. Significant progress has been made with surgery, adjuvant chemotherapy and radiotherapy, However, despite advances in therapy, the five-year survival remained fairly static over the past decade. New measures are urgently needed. Gene therapy represents a rational new approach to cancer therapy, which could provide an adjunct to conventional treatment. To maximize the tumor cell killing effect, combining Chinese traditional medicine and gene therapy may be an approach.Chrysanthemum morifolium Ramat contains many kinds of flavone compounds. These compounds have many biological activities, the most important activity is their anti-cancer function. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF gene superfamily that can induce apoptosis through engagement of death receptors. These receptors include two pro-apoptotic death receptors and threeanti-apoptotic decoys receptors. This protein has generated tremendous excitement as a potential tumor-specific cancer therapeutic because, both in a stable soluble form and membrane bound form, it selectively induces apoptosis in many transformed cells but not in normal cells.The goal of this experiment is to find a new way to treat caner by combing Chinese traditional medicine and gene therapy, we evaluate the therapeutic efficiency of Chrysanthemum morifolium Ramat, the therapeutic efficiency of Chrysanthemum morifolium Ramat combined with TRAIL gene on human colon cancer cell line DLD1.Materials and methodsMaterialCell lines: 293 cell, human colon cell line DLD1 Adenoviral vectors: Ad/hTERT-gTRAIL and Ad/CMV-GFP Others: experiment apparatus, materials for cell cultureMethodsl)The expansion, purification, titration and quality analysis of all the vectors: Ad/hTERT-gTRAIL and Ad/CMV-GFP are expanded in 293 cells, purified by CsCl banding, titrated by mensurating the OD value of A260.2) The expression of GFP/TRAIL gene: 1 X 105DLD1 cell were inoculated in 6-well plate, 24 hours after inoculation, cells were treated with Ad/hTERT-gTRAIL, Ad/CMV-GFP, TRAIL/Chrysanthemum morifolium Ramat combination, Ad/CMV-GFP/Chrysanthemum morifolium Ramat combination and PBS; 48 hours after treatment, GFP/TRAIL gene expression was determined by Flow Cytometry.3) Cell viability assay: 5X 103 DLD1 cell were inoculated in 96-well plate, set up 4 parallel team, 24 hours after inoculation, cells were treated with the following six groups: TRAIL gene from the hTERT promoter(Ad/hTERT-gTRAIL), Chrysanthemum morifolium Ramat, Ad/CMV-GFP , Ad/CMV-GFP/Chrysanthemum morifolium Ramat combination, TRAIL/Chrysanthemum morifolium Ramat combination and PBS(Blank control). 48 hours after treatment, the cell viabilities were determined by MTT assay.4)Apoptosis assay: 1 X 105DLD1 cell were inoculated in 6-well plate, 24 hours after inoculation, Cells were treated with the following six groups: TRAIL gene from the hTERT promoter (Ad/hTERT-gTRAIL), Chrysanthemum morifolium Ramat, Ad/CMV-GFP, Ad/CMV-GFP/Chrysanthemum morifolium Ramat combination, TRAIL/Chrysanthemum morifolium Ramat combination and PBS(Blank control). The cell morphology was checked under an inverted microscope every day after treatment, and 48 hours later, cells were collected and labeled with propidium iodide (PI), subjected to Flow Cytometry. 4) Statistics analysis: SPSS 10.0 software was used in the statistic analysis, the significance was claimed when P<0.05.Results1. Virus titers determined by A260 was 1 X 10~11 particles/ml after expand and purification.2. Gene expression: FACS showed 82.53% and 83.2% GFP expression after treatment of Ad/hTERT-gTRAIL and Ad/CMV-GFP. It also sho... |
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