2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644428摘要：B 細胞在移植排斥的免疫反應中扮演重要的角色，B 細胞可以分化為漿細胞進而產生抗體，是抗體性排斥居中的重要媒介。而且，對人體器官移植而言，HLA 抗體與慢性移植腎病變有關。最近，我們發現去除B 細胞可以減少中心記憶T 細胞的比率而且抑制高敏感性移植之記憶T 細胞反應；該論文已發表於？。目前，我們正進行前膽性之臨床試驗，探討去除B 細胞是否有助於降低急性排斥率與改善腎功能。我們認為進一步以動物實驗研究去除B 細胞抑制記憶T 細胞之免疫機制可以進一步改善移植病患之預後。先期研究中，我們發現促進凋亡之Bim 基因表現在去除B 細胞後有增加現象，顯示去除B 細胞可能透過促進T 細胞之凋亡抑制T 細胞之記憶反應，因此，我們提出假說：B 細胞藉由促進T 細胞凋亡抑制T 細胞記憶反應。第一年：我們先研究去除B 細胞與否在混合淋巴球試驗中T 細胞增殖，在不同時間點細胞激素(IL-2, IL-7 and IFN-γ)產生與接受體IL-2Rα(CD25)和IL-7Rα(CD127)表現之差別。第二年：計畫分析在不同時間點去除B 細胞與否對Bcl-2，Bim 在Q-PCR 與西方點墨法上之表現之差異，並且進一步以流式細胞儀分析T 細胞在刺激後，凋亡的現象是否受去除B 細胞與否影響。另外，為進一步探討細胞激素對T 細胞凋亡之影響，計畫在混合淋巴球試驗中加入細胞激素抗體(anti-IL-2, anti-IL-7 and anti-IFN-γ)然後再次分析Bcl-2 與Bim 之基因表現。第三年：我們以心移植模型探討去除B 細胞之後移植心臟中是否存在比較多的凋亡T 細胞，以免疫化學染色和流式細胞儀分析心移植浸潤細胞凋亡相關分子的表現。研究成果預期可以進一步確認去除B 細胞在移植後免疫抑制策略上的定位，而且可以為臨床器官移植的病人找尋新的免疫調控方式，改善移植成果。<br> Abstract: B cells play an important role in allogeneic immune responses. B cells, whichdifferentiate into plasma cells to produce antibodies, are central to antibody-mediatedallograft rejection. And, for human renal allograft transplantation, anti-HLA antibodieswere found to be associated with chronic allograft nephropathy and graft failure. Recentlywe found that B-cell depletion reduced the percentage of central memory T cells in thespleen and inhibited the memory T-cell allogeneic immune responses in sensitized recipients;the results have been published in Transplant Immunology 2009. And, we are nowconducting a prospective randomized clinical trial in renal transplant patients looking at ifB-cell depletion would reduce acute rejection and improve renal function (NSC:97-2314-B-002-175-MY3). We believed that further investigation into the mechanismsresponsible for the suppression of memory T-cell immune response by B cell depletion inanimals would help to improve clinical outcomes of transplant patients.Preliminarily, we found that the gene expression of proapoptotic Bim increased when Tcells received allogeneic stimulation in absence of B cells, suggesting that B cell depletioncould be lessening T cell memory response by enhancement of T cell apoptosis. Accordingly,we hypothesize that B cell depletion lessens T cell memory responses by enhancement of Tcell apoptosis.In the first year, we plan to study the proliferation, cytokine production and receptors ofT cells in the absence of B cells or not. Thymidine incorporation, ELISA and Q-PCR ofcytokines (IL-2, IL-7 and IFN-γ) and flow cytometric analysis of IL-2Rα(CD25) andIL-7Rα(CD127) will be conducted after mixed lymphocyte cultures.In the second year, we will focus on the apoptosis-related gene and expression in vitro.Q-PCR of Bcl-2 and Bim will be measured on 48, 96 and 168 hours in mixed lymphocytecultures with or without B cell depletion. Western blots of Bcl-2 and Bim will also beperformed at various time points to confirm the results of Q-PCR. Flow cytometricanalysis fo annexin-V, 7-AAD and caspase-3 will be conducted to see if more apoptosishappened when B cells depleted. And, to further characterized the role of cytokines (IL-2,IL-7 and IFN-γ) in allogeneic T cell apoptosis, blocking antibodies (anti-IL-2, anti-IL-7 andanti-IFN-γ) will be added to the cultures systems, and assays for apoptosis and Q-PCR ofBcl-2 and Bim conducted again.In the third year, we would like to demonstrate that more apoptosis happens in the hearttransplant grafts when B cells depleted in the recipients. A heart transplant model with Bcell depletion will be set up using ex vivo B cell depletion and adoptive transfer to SCIDmouse. C57BL/6 heart grafts will be removed from the recipients at various time points forimmunohistochemistry study of Bcl-2 and Bim. Graft-infiltrating cells will also beharvested for flow cytometric analysis of apoptosis markers including annexin-V, 7-AADand caspase-3.B細胞細胞凋亡B cellsapoptosis