| I diabetes (T1D) is also known as insulin-dependent diabetes mellitus, is a chronic organ-specific autoimmune diseases. When the body is due to genetic variation, virus infection and other factors induce the immune system disorders, abnormal autoreactive T cell amplification, a large number of mononuclear cell infiltration of islet tissue occurs, causing β cell destruction, persistent elevated blood sugar will eventually lead to the formation diabetes. Which can maintain islet antigen-specific CTL inflammation, and is responsible for directly kill P cells, which is particularly important in T1D. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) was mainly expressed in pancreatic βcells, and for IGRP206-214epitope-specific CTL cells to accelerate the development of T1D has an important role, has been reported by toxin tetramer tetramer conjugated toxin saporin (SAP) prepared by the method can selectively remove non-obese diabetic (NOD) mice IGRP206-214-specific CTL, delaying the occurrence of T1D.MHC-I tetramer technology has many advantages, but there are not part of their stability and specific CTL and T-cell receptor (TCR) lower affinity disadvantages, our laboratory we introduce a single chain trimer body technology (SCT) technology to optimize successfully constructed IGRP206-214dtSCT prokaryotic expression vector, and prepared IGRP206-214dtSCT tetramer. In this experiment, as the basis of our attempts prepared saporin conjugated IGRP206-214dtSCT tetramer and study its effect on disease progression in NOD mice T1D.We first induced IGRP206-214dtSCT prokaryotic expression vector expressed protein, after inclusion bodies washing, protein purification, refolding, ultrafiltration interception, biotinylated protein monomers obtained, then the resulting IGRP206-214dtSCT monomer (IGRP-SCT) and conjugated saporin streptavidin-biotin conjugates were prepared by incubating4.5:1saporin of IGRP206-214dtSCT tetramer (IGRP-SAP). We then applied the fluorescent conjugated PE prepared for IGRP206-214dtSCT tetramer (IGRP-PE) as a detection tool to analyze NOD IGRP-SCT, IGRP206-214dtSCT tetramer (IGRP-Tetramer) and IGRP-SAP vitro mouse lymphocytes affect IGRP206-214specific CTL and its possible mechanism. Finally, to study its ability to clear IGRP206-214specific CTL in vivo, and the impact on the process of T1D in NOD mice with IGRP-SAP intravenous injection of8-week-old NOD mice.Detected by the FACS, the IGRP-Tetramer we have prepared have more capacity than soluble IGRP-SCT reduced the frequence of IGRP206-214specific CTL in vitro, while the solid phase can be increased IGRP-SCT IGRP206-214specific in vitro frequency of CTL, and its ability to slightly IGRP206-214peptide. Further IGRP-SAP can vitro induced reduced approximately80%IGRP206-214specific CTL, NOD mice were injected continuously for three weeks after IGRP-SAP, IGRP206-214specific CTL in vivo detection rate has dropped to about75%by continuous glucose monitoring, we found that glucose IGRP-SAP group compared to NOD mice significantly decreased in the PBS group at24weeks of age, the incidence rate of60%PBS group dropped to10%.Experimental results show that we successfully prepared the IGRP-SAP, and be able to clear the NOD mouse IGRP206-214specific CTL in vivo, but also in vivo inhibition of proliferation of CTL. Cleared IGRP206-214specific CTL could significantly delay the occurrence of T1D, and reduce the incidence of NOD mice. This topic after this lay the foundation against InsB15.23specific Targeted cleared CTL, but also for the NOD mouse TID-related T cell subsets analysis provided the conditions. |
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