| Background and objective:T1DM is perceived as a T lymphocyte mediated auto-immune diabete mellitus,which is characterized by selective loss of insulin-producingβ-cells in the pancrea- tic islets in genetically susceptible subjects. Clinical T1DM represents the end-stage of insulitis, and it has been estimated that at the time of diagnosis about 80% of theβ-cells are destroyed . So the diagnosis and prevention of T1DM in the early time seems very important . A series auto-antigens like insulin , GAD , IA-2 and ZnT8 are perceived as the target antigens in initiating the auto-immune reaction . At the same time,some cytokines , chemotactic factors and inflammation mediators also participate in this immune process . Type 1 T helper cells secrete IFN-γinduced the development of T1DM , while , in the opposite side , Type 2 T helper cells which secrete IL-10 induces a protective effect in the development of T1DM . some researchers believed that insulin/proinsulin, especially insulin B:9–23 peptide, is mostly likely a primary autoantigen to initiate immune-mediated diabetes of the NOD mouse. To invest wether the auto-antigens can induce immune tolerance in NOD mice ,Various antigen-specific immunotherapies using insulin and insulin peptide have been evaluated using the NOD mouse model. Such as intranasal or subcutaneous administration of insulin B:9–23 peptides or an altered insulin B:9–23 delays diabetes development . All of the researches were draw assistance of immunological adjuvant which can enhance the antigen immunogenicity , perhaps it is because that antigen alone can not presented well by antigen presenting cells . In our studies , we view from the point of interaction between DC-T cells . Using insulin B9-23peptide load DCs in vitro , approaching the effect and the possible mechanism in inducing immune tolerance .DCs are the most important professional antigen presenting cells in vivo , and act as a important role in inducing the development of T1DM . Meanwhile , DCs also have a center role in both centra and periphery immune tolerance . So DCs were widely used in the treatment of auto-immune disease . Under the steady state , DCs expressing low level co-stimulators such as CD80 , CD86 , CD40 ,have a strong capacity in uptaking the antigen but a weak capacity in presenting . Three signals are needed in the process of T cell activation : The first signal involves the presentation of antigen on the surface of an MHC class II molecule, which facilitates T cell recognition of the cognate antigen though the TCR. In order for antigen-specific T cells to become activated and this population to expand in number, a second signal must be generated through the interaction of adhesion and costimulatory molecules present on the APC, such as CD80 and CD86, with CD28 on the surface of T cells. The third signal is the secretion of cytokines by APCs, which directs the differentiation of activated antigen-specific lymphocytes into an effector T cell subtype. If the second signal were inhibited , the na?ve T cells will be induced into apoptosis or anergy . As we all know that the CD80 , CD86 and CD40 are the most important co-stimulator on the surface of DCs . In our study , we use RNAi technology to inhibite the expressing of CD80 , CD86 and CD40 , then loaded insulin B9-23 peptide in vitro , preparing tolerogenic DC vaccine , observing the effect in stimulating T cells , approaching the effect and mechanism in the treatment of T1DM , providing experiment accordance of the early prevention and treatment of T1DM .Methods:1. The siRNA of costimulatory molecules CD80,CD86,CD40 were designed and synthesized . Then they were transfected into dendritic cells by conjuncted with lipofectamine 2000.2. Dendritic cells were obtained by isolating the bone marrow from ICR mice. Cells were cultured in 24-well plates at 2×106cells sml in 1 ml CM(RPMI-1640, supplemented with 10% foetal calf serum,10 ng aml GM-CSF and 10 ng fml IL-4 ). At day 2 and day 4,fresh cytokines and CM were added and the cells were re-incubated. At day 6, non- and loose-adherent cells were harvested and cultured in 24-well plates at 2×105 cells tml in 500 ul CM supplemented with GM-CSF and IL-4 which provided 50–70% con?uency of the cells as required by the manu-factures prior to the transfection. After 24 hours incubation , cells were devided into four groups , named as groupA(add CD80/86/40siRNA and lipofectamine 2000),groupB(add negative control siRNA and lipofectamine 2000),group C(add nothing),group D(add FAM-conjugated siRNA and lipofectamine 2000 ) . At day 8 ,all the four group dendritic cell were harvested .3. Using the confocal microscopy to calculate the efficiency of transfection.4. Using flow cytometry to analyse the expression of CD11c,CD80,CD86,CD40 and the efficiency of transfection .5. Using rt-PCR to detec the expression of CD80,CD86,CD40 .6. Mixed lymphocyte reaction: 4×104 DC of group A,B,C treated with mitomycin C and pulsed with insulin B9-23 peptide were cultured together with 2×105 T cells from mesenteric lymph nodes of ICR mice and the cells were cultured for 72 hours including 18 h incorporation of 0.5 lCi dwell[3H] thymidine for the measurement of proliferation.7. Using ELISA to analyse the IL-10 and IFN-γsecreted by T cells .Results :1. Under the confocal microscopy , about 80% cells were seen with green fluorescence .2. Flow cytometry detected that the efficiency of transfection was 79.92%,the CD11c expression of four groups were all above 60%. compared with groupB and groupC,the CD80/86/40 expression of groupA were lower ,but there is no significant difference .3. rt-PCR displayed that the CD80/86/40expression of groupA were down regulated.4. Mixed lymphocyte reaction: The CPM value of groupA ,groupB, groupC is 2424.67±169.36,7484.33±133.44,7735.33±539.87 in apart . And dendritic cells of groupA have the weak ability to stimulate the T cells . (p<0.01)5. The analyse of cytokine:The IL-10 secreted by Tcells of groupA,groupB,groupC is 522.72±6.92,483.40±4.70,483.91±6.82 apart,and the IFN-γ(pg/ml)is 1654.95±39.79,1971.76±62.85,1936.87±61.96 apart,compared with groupB and groupC , the Tcells from group A secreted more IL-10 and less IFN-γ, the difference has statistic significance (p<0.01).Conclusion:Selective gene silencing using RNA interference can effectively depress the expression of costimulatory molecules CD80,CD86,CD40 of dendritic cells derived from mouse bone marrow. These dendritic cells loaded by insulinB9-23 peptide have weaker ability in stimulating T lymphocytes proliferating , induce na?ve T lymphocytes polarized into Th2 cells ,secrete more IL-10 and less IFN-γ.This study indicate that insulin B9-23 peptide load toleragenic dendritic cells in vitro can induce immune tolerance , by inducing T cells anergy and polarize na?ve T cells into Th2 cells.These effects have big significance in early prevention of autoimmune diabetes. |
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