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<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> | <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> | <h3 class="ar-title"><span class="dg"> </span>RESULTS</h3> | ||
− | < | + | <h2><br>1.The construction and expressing validation of SynNotch and CAR system</h2> |
<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> | <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> | ||
<br> | <br> | ||
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<h4 align=middle>Figure8. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation</h4> | <h4 align=middle>Figure8. The expression of SynNotch and CAR system in Flag-FOXP3-Jurkat cell line introduced by lentiviral transfection and electroporation</h4> | ||
</div> | </div> | ||
− | < | + | <h2><br>2.The functioning validation of SynNotch and CAR system</h2> |
<br> | <br> | ||
<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 9), 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 9), indicating that the SynNotch system stabilized FOXP3 in Treg cells with the presence of IL-6.</h4> |
Revision as of 02:47, 2 November 2017