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Induction of Allograft Tolerance by NF-κB Oligodeoxyribonucleotide Decoy-Pretreated Donor and Recipient Dendritic Cells Loaded with Donor-Derived Apoptotic Cells

Author: XuDongLiang
Tutor: TangXiaoDa
School: Fudan University
Course: Surgery
Keywords: Dendritic cells Nuclear factor -KB Decoy Mice Heart transplant Immune tolerance Apoptosis UVB
CLC: R392
Type: PhD thesis
Year: 2004
Downloads: 158
Quote: 0
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Abstract


Objective: to establish cultured in vitro amplification of mouse bone marrow-derived dendritic cells (dendritic cells, DC), observed oligodeoxynucleotide decoy nuclear factor-κB (NF-κB ODN Decoy) for DC mature, and biological characteristics of Immunology, 2, ultraviolet B (UVB) irradiation induced splenic apoptosis observed NF-κB ODN Decoy ability to handle DC engulfed with apoptotic spleen cells (Apo-SCs) as well as DC biological changes in the immunological characteristics swallowed explore DC cross-presentation and cross-sensitization mechanism of action, and to explore the induced cross-tolerance approach. 3, establish allogeneic mice heterotopic cardiac transplantation model, observed or / and phagocytosis of apoptotic cells of the donor recipients before transplantation, infusion of NF-κB ODN Decoy process for DC survival of mice transplanted heart donor and recipient, explore the process of NF-κB ODN Decoy possible mechanisms of transplantation tolerance induced by DC. Methods: 1, in vitro isolation of bone marrow precursor cells, recombinant mouse granulocyte - macrophage colony stimulating factor (rmGM-CSF) stimulation, tactics screened, cultured and expanded in mouse bone marrow-derived DC (BM-DC). Application-specific NF-κB ODN Decoy handle DC observed DC morphology, expression of cell surface molecules, phagocytosis, and the same kinds of mixed lymphocyte reaction changes, understand the impact of the NF-κB ODN Decoy DC maturation and biological immunological characteristics. 2, UVB irradiation, spleen cells induced apoptosis, and induction of apoptosis detection by flow cytometry (FCM); coculture with apoptotic spleen cells (Apo-SCs) and DC with NF- κB ODN Decoy pretreatment DC (Decoy Apo-SCs DC), application of FCM and fluorescence microscopy engulfed with apoptotic cells the ability to understand the DC. The changes in the expression of surface molecules by FCM DC, understand The Decoy Apo-SCs DC to stimulate allogeneic T cell proliferation ability, and with the Decoy Apo-SCs DC pre-sensitized mice line again mixed lymphocyte reaction, through the initial mixed lymphocyte reaction observed Decoy Apo-SCs DC cross-sensitization and cross-tolerance ability of allogeneic T cells. 3, celiac double vascular side anastomosis to establish the same kind of mouse heterotopic cardiac transplantation model, donor BALB / c mice, C57BL / 6 mice were used as recipients, seven days before the transplant surgery portal vein infusion DC (2 × 10 ~ 6cells) treated by the method pretreatment recipient mice, depending on the infusion of DC will be the real NF · KBOONOecoy processing for cross dendritic cells induced a guide for Free degree resistant to the history of the real face Chinese Summary inspection mice were divided into seven groups: ① control group (Colltrol): a recipient via the portal vein 7 days before surgery simple the infusion o.Zml phosphate buffered solution (PBS); Lee) DC group: infusion without NF KB ODN Deeoy source of donor BM DC; ③ Deeoy DC group: the source of donor BM infusion of NF-KB ODN neeoy processing oC; ④ of Apo the ses DC group: lose should i: NF the one KB OnN Deeoy unprocessed phagocytosis of the same kinds of Apo-SCs source of recipient BM-DC; ⑤ Deeoy of Apo sCs De the group: the infusion of NF-KB oDN Deeoy processing swallowed microphone donor recipients De Apo an SCs; the ⑥ joint infusion group: United infusion of NF-KB ODN Decoy treated donor derived DC and phagocytic receptor sources De; Apo a ses donor the ⑦ third for the peer group (\Group 6. survival time of mice transplanted heart and histopathological changes, using semi-quantitative RT-PCR method to detect the mice transplanted heart tissue cytokines IL-2, a Hell of IIJ-10 and IFN- mRNA levels.: l: mGM CSF stimulation, can be cultured from mouse bone marrow amplification l} of a large number of DC, BM-DC is obtained from about 10 2 per mouse. another 1. \can meet the needs of the experiment. DC, NF-KB oDN Decoy processing performance in morphologically immature state, cell surface costimulatory molecules CDSO, CD86, CO40 expression was significantly lower, and inhibition of IL-12 secretion hindered the DC maturation, which kinds Yang hinder the role can not be reversed lipopolysaccharide (LPS) stimulation. Mixed lymphocyte reaction of NF a KB ODN Decoy inhibits DC to stimulate the same kind of lymphocyte proliferative responses and T cells secrete Thl type cytokines. UvB irradiation effectively induced the spleen cell withered Guang. FcM detection, uvB (20omJ/cmZ) cultured for 18 hours after irradiation, nearly 90% of the apoptotic spleen cells can be obtained. DC can be effectively swallowed with spleen cells of apoptosis. Withered spleen cells with DC incubated together for 48 hours, FCM detection and fluorescence microscopy results show that DC can be effectively swallowed with spleen cells of apoptosis. NF-KB ODN Decoy inhibits DC swallow the same kinds of apoptotic spleen cells mature. DC with the same kinds of apoptotic spleen cells incubated together stimulate the mature DC phenotype; NF a KB ODN Decoy pre-treated DC inhibit the phagocytosis with withered one death caused by the spleen cells of DC maturation. The initial mixed lymphocyte reaction Decoy Apo an Scs DC can significantly inhibit the proliferation of allogeneic T cells in vitro reaction; once again mixed lymphocyte reaction results show Decoy Ap. A Scs Dc allogeneic T cells can induce antigen-specific immune hyporesponsiveness and cross-tolerance. Successfully established a stable allogeneic mice heterotopic cardiac transplantation model, syngeneic mice (C57BL / 6 a C57BL / 6) cardiac allograft survival! 'Division gt; 100 days. Groups the allogeneic mice transplanted heart off (B eight LB / c * C57BL / 6), the average survival time (MST): control group to 7.1 days, DC Davis!} 8.4, Deeoy DC group 20 days, Apo a SCsDC to [l'5 .1 days, Deeoy Apo one SCs De wholly-11 33.3 days 65.1 days, combined infusion group, the third NF a KBOONOecoy hoot reasonable for cross dendritic cells induced free attack the real face of resistance to cross research brother Chinese Abstract donor group (3rd party, e3llzlleJ * e57RLz6) 5.7 Tian. Deeoy De group De group difference was statistically significant (P = 0.0003; Deeoy of Apo sCs DC group Apo a SCs DC group compared to the group with Deeoy DC significant difference (p == 0.0003 = 0.0001); the longest combined infusion graft survival, there are significant with the other group, the difference was statistically significant 'with the control group, P = 0.000: DC group p 0.0002: with the Deeoy DC group P = 0.0002: and Apo the SCs DC group p = 0.0001: with Deeoy Apo SCs DC group p = 0.0086: the third donor group, P = 0.0003). 9 detected in mice transplanted heart group?

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