Exploring the Mechanism of Immunological Responses in Liver Transplantation–From Rejection to Tolerance in DA-PVG Model

在這篇論文中，我們建立了一個自發性耐受的模式，兩種不同品系的大鼠DA與PVG在進行異體肝臟移植後，可以在不外加任何免疫抑制的處理下，自發性的對外來器官產生耐受的反應。利用功能性蛋白質體學分析在異體肝臟移植中有不同表現量的血清蛋白質，發現在這些不同表現量的蛋白質中，包括血紅素結合蛋白 (haptoglobin)以及激肽原 (kininogen)皆有報告提出具有抑制淋巴球細胞的增生能力，而因此可能降低細胞調控的排斥反應。根據這些不同表現量的蛋白質，透過生物資訊分析統計上有顯著差異的網絡 (network)，發現介白質6 (Interleukin-6)可能參與這個自發性耐受的模式。介白質6是一種具有肝臟保護能力的白血球細胞生長激素，它可以促進肝臟的再生、減少肝臟的受損以及提高具有免疫抑制能力的蛋白質例如血紅素結合蛋白的表現量。有趣的是，在這個模式中，介白質6表現的時間點正好是器官移植排斥最嚴重的時期，因此我們推測介白質6可能在這個自發性耐受上扮演關鍵的角色。此外，介白質6可以提高岩澡糖基化 (fucosylation)調控基因的表現，也支持我們所觀察到在模式中，血清蛋白質的岩澡糖基化程度有上升的情形，但是岩澡糖基化在這個模式中可能的相關功能目前尚不清楚。這些觀察到的現象暗示介白質6在異體移植中扮演著保護的角色。In this report, we have established a spontaneous tolerance model in which rats could develop spontaneous tolerance without any immunosuppressive treatments after orthotopic liver transplantion between DA (RT-1a) and PVG (RT-1c) rats. Functional proteomics analysis was introduced to investigate the differently expressed proteins involved in allograft liver transplantation. Among these different-expressed proteins, both haptoglobin and kininogen were reported to inhibit the proliferation of lymphocytes, suggesting a possible role of these two proteins in down-regulate cell-mediated graft rejection. Bioinformatics analysis of the statistically significant networks based on these differently expressed proteins indicated that the IL-6 signaling pathway might be involved in this model. IL-6 is a hepatoprotective cytokine that promotes liver regeneration, reduces liver injury and up-regulates the immune- suppressive proteins such as haptoglobin. Interestingly in this model, the highest expression of IL-6 occurred at the most severe stage of allograft rejection; thus we hypothesize that IL-6 might be the key contributor to spontaneous tolerance. Furthermore, it has been previously shown that IL-6 can also up-regulate the fucosylation regulatory genes, which supports our observation of an increase of fucosylation levels in serum protein; yet the related functions of fucosylation in this model remained unclear. These observations suggest that IL-6 plays a protective role in the spontaneous tolerance DA/PVG OLT model.Acknowledgements 2bstract (Chinese) 4bstract (English) 5bbreviations 10ists of Figures 12ists of Figures 13. Introduction 14.1. Organ transplantation 14.2. Transplantation rejection and immunosuppressive therapy 15.3. Spontaneous tolerance orthotopic liver transplantation 16.4. Functional proteomics analysis 17.5. Investigation of IL-6 related responses in DA/PVG OLT model 19. Materials and Methods 21.1. Orthotopic liver transplantation 21.2. Sample preparation 21.3. Two-dimensional gel electrophoresis (2DE) 21.4. Two-dimensional difference gel electrophoresis (2D-DIGE) 22.5. In-gel digestion for protein identification 23.6. Mass spectrometry analysis 24.7. Bioinformatics analysis 25.8. Enzyme-linked immunosorbent assay (ELISA) of IL-6 26.9. Western blot assay 26.10. Lectin blot assay 27. Results 29.1. Analysis of protein expression in DA/PVG OLT model 29.2. Identification of different-expressed serum protein 29.3. Functional networks analysis of DA/PVG OLT model 30.4. ELISA Quantification of IL-6 30.5. The expression time course of haptoglobin,α1-antitrypsin, and ceruloplasmin 31.6. Lectin blot analysis the fucosylation of serum protein 31. Discussion 33.1. Functional proteomics analysis in DA/PVG OLT model 33.2. Potential immunosuppressive proteins 33.3. Bioinformatics network analysis in DA/PVG OLT model 34.4. Il-6 signaling pathway 35.5. Role of IL-6 in organ transplantation 36.6. IL-6 regulated proteins 37.7. The expression time course of IL-6 and IL-6 regulated proteins 38.8. Glycan modification in DA/PVG OLT model 39.9. Conculsion 40. Reference 42. Figures 51. Tables 6