| The development of cancer is complex and disorderly processes. The mechanisms and causes of cancers are very complex, involving innate heredity and acquired dispositions. Because of its complexity and heterogenity, the current cancer therapy is still a challenging problem to global medicine. With the development of human medical and biological sciences, many more modern methods are applied to the treatments of cancers. Because of the shortcomings on traditional cancer therapies, the multi-target treatment for cancer has become the new hotspots in cancer researches.TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) is TNF-a related cytokines family member. For its nontoxicity for normal cells and special toxicity for tumor cells, TRAIL has become the most potential medicine for cancer treatment. However, the cost of manufacture of TRAIL its transportation and storage is very huge, so finding the small molecular analog of TRAIL is very important for cancer therapy, but it is a challenge to find the functional analog of TRAIL from the natural phytochemicals.A molecular fluorescence labeling technique created by Canon in USA provided a new approach to resolve this problem; they use GFP-marked β-arresting to detect G-protein-coupled receptors activation. Also, we can use this technology to mark Procaspase-8 with GFP to screen small molecules from the natural phytochemical extracts which have the same effects as TRAIL. The construction of stable cell lines containing GFP-labeled Procaspase-8 is essential for which we can selectly screen out small molecular analogs of TRAILYunnan is rich in natural medicinal resources. The technology of Receptor-Effector Fluorescence Imaging (REFI) provides us a reliable tool for the discovery and selectively screening of TRAIL mimic analogs from natual resources.In order to avoid the excessive expression of Procaspase-8 which can induce cell apoptosis, GFP is constructed behind prc8DED1 to replace the Caspase-8 enzymatic domain of Procaspase-8, so that the constructed cells will not automatically apoptosize or be induced to apoptosis by TRAILThe cell model has been successfully constructed. The aims of my research are first to work out the best experimental conditions in which the REFI effects of TRAIL can be reliably and consistenly determined in term of reaction time, TRAIL concentrations and cell conditions.Secondly, using CRISPR/Cas9 editing technique to knockdown the endogenous Procaspase-8, so that the Prc8DED1-GFP can bind to DR4/5 more effectively without the interference from competitive binding of endogenous Procaspase-8.Experimental contents and results:1. Cell lines U2OS-Prc8DED1-GFP was successfully constructed by molecular biology methods. Thes cells were cultivated and grew in 96-well plates to 80% confluence, and treated with TRAIL at the gradients of concentrations at varible time points to identify wether the cell line U2OS-Prc8DED1-GFP is established. When the cell was treated with TRAIL, the GFP fluorescent spots began to assembled under fluorescence microscope observation. After about 30 min TRAIL treatment, the GFP green spots recovered to its original state, indicating that the cells can surely respond to TRAIL.2. In order to further optimize the cell line U2OS-Prc8DED1-GFP to avoid the competitive binding of the endogenous Procaspase-8 to receptors DR4/5, the new gene editing technology CRISPR/Cas9 was used to knockdown the endogenous Procaspase-8. The Donor-DNA and Guide-RNA were constructed, and transfected into the U2OS-Prc8DED1-GFP cells, the monoclonal cells with red light were cloned.Conclusion:The cell line U2OS-Prc8DED1-GFP has been preliminarily confirmed to respond to TRAIL treatment. CRISPR, and Cas9 knockout plasmids have been constructed, which were successfully transfected into U2OS-Prc8DED1-GFP, and we are in process in establishing and charterizing the cell lines. |
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