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<center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> | <center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> | ||
− | <h4 align=middle> | + | <h4 align=middle>Figure6. Design of SynNotch system and CAR system</h4> |
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− | <h4><br> | + | <h3 class="ar-title"><span class="dg"> </span>CAR System</h3> |
+ | <h4><br>Next, we design a sharp spear for Treg. We choose the B lymphocyte with CD20 high expression as our target for Treg, so we introduce a CAR system into Treg to let it specifically recognize the CD20 antigen at the surface of B lymphocytes in order to achieve anti-inflammatory function. As for the design of our CAR system, we use the scFv fragment of CD20 monoclonal antibody as the extracellular fraction because it can recognize and bind with CD20 on B lymphocyte’s surface accurately. We then choose a CD3Z sequence as the stimulatory signal and two 4-1-BB sequence as the co-stimulatory signal to make sure the signal can be delivered into the cell at a high level, thus activating an effective response of Treg. We attach a red fluorescent protein mCherry to the end of the CAR fusion protein as a report signal for a more convenient detection. By designing this CAR system we equip our Treg with a spear, which can reduce the inflammation level in RA patients.</h4> | ||
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+ | <h4><br>Based on our design, we name our engineered Treg as “Human Engineered Anti-Autoimmune-Diseases Regulatory T cell (HEAD-Treg) System”. We hope it can serve as a mighty warrior, leading the immune system to rebalance itself, to conquer the disease and to reestablish the patient’s health.</h4> | ||
+ | <div> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2017/f/f4/T--CPU_CHINA--results_fig1.png" width = "800"></center> | ||
+ | <h4 align=middle>Figure7. HEAD-Treg System</h4> | ||
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+ | <h3 class="ar-title"><span class="dg"> </span>Summary of Mathematical Models</h3> | ||
+ | <h4><br>In order to further evaluate the clinical value of our system and provide a theoretical reference for the next in vivo and preclinical experiments, we also establish a systematic mathematical model for the local and overall immune environment of rheumatoid arthritis (See the Model section for details). Thus, we have a theoretical explanation of the relationships among the cytokines, different cell subpopulations and rheumatoid arthritis, which lays a solid foundation for further improvement of our research ideas regarding the subsequent in vivo and preclinical experiments</h4> | ||
+ | <h3 class="ar-title"><span class="dg"> </span>RESULTS</h3> | ||
+ | <h4><br>1.The construction and expressing validation of SynNotch and CAR system</h4> | ||
+ | <h4><br>To engineer our regulatory T cells, we designed a three-plasmid expressing system for genes of SynNotch and CAR. We chose PLVX-IRES-Puro, PLVX-IRES-Neo and pcDNA3.1 as backbones for the plasmid vectors that carry the SynNotch fusion protein gene, the CAR-CD20 fusion protein gene and the UAS-USP7-promoter-USP7 sequence individually (these three genes were synthesized and connected to their vectors by Genscript). Find more details about the design of the fusion protein and the plasmid vector in Parts section.</h4> | ||
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+ | <h4><br>To test the feasibility of transfecting multiple plasmids into Treg cells, we acquired the Flag-FOXP3-Jurkat cell line from Shanghai Institute of Immunology, Medical College, Shanghai Jiao Tong University. As a stable transfection strain with high expression of Flag-FOXP3 obtained by transfecting Flag-FOXP3 fusion protein into Jurkat T cells, it is a decent model to simulate the Treg status in human body. In our experiment, we introduced our three-plasmid expressing system into the Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of both SynNotch and CAR system in Flag-FOXP3-Jurkat cells were confirmed by western blot and quantitative real-time PCR (Figure 1).</h4> | ||
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<h4><br>To test SynNotch’s stabilization function on FOXP3 in Treg cells under inflammatory conditions, inflammatory factor IL-6 was added into the culture medium to simulate the microenvironment in RA patients, then western blot and quantitative real-time PCR were performed. Without IL-17A, the expression of FOXP3 was significantly reduced compared to normal one due to the inactivation of SynNotch. However, with the addition of IL-17A, the FOXP3 level was greatly uplifted (Figure 2), indicating that the SynNotch system stabilized FOXP3 in Treg cells with the presence of IL-6. </h4> | <h4><br>To test SynNotch’s stabilization function on FOXP3 in Treg cells under inflammatory conditions, inflammatory factor IL-6 was added into the culture medium to simulate the microenvironment in RA patients, then western blot and quantitative real-time PCR were performed. Without IL-17A, the expression of FOXP3 was significantly reduced compared to normal one due to the inactivation of SynNotch. However, with the addition of IL-17A, the FOXP3 level was greatly uplifted (Figure 2), indicating that the SynNotch system stabilized FOXP3 in Treg cells with the presence of IL-6. </h4> | ||
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Revision as of 17:58, 1 November 2017