2010-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/696357摘要：活性氧化物系在adaptive immune response 中扮演一個重要的角色，在抗原辨識之過程中，細胞內之活性氧化物會造成抗原呈現細胞產生造成adaptive immune response。在傳統抗原呈現路徑，一般認為內生性抗原會經由MHC-I，而外生性抗則會經由MHC-II 之抗原呈現路徑，然而外生性抗原亦會經由MHC-I，導致CD8+ T 細胞毒殺效應(cytotoxic lymphocytesreaction)，亦即所謂的cross-presentation。在此計劃中，我們針對本實驗室過去在研究抗原遞送實驗的一些結果提出的一個假設， 擬你探討活性氧化物是否造成專一性抗原cross-presentation 的一個必要條件。為測試此一假設，我們擬以ovalbumin (OVA)作為模式抗原， 再以疫苗佐劑處理抗原呈現細胞(antigen-presenting cells) ， 包括樹突細胞(bonemarrow-derived dendritic cells, DCs) 與巨噬細胞 (macrophages)等。同時擬在細胞以佐劑處理前，先以N-acetyl-L-cysteine 等數種之抗氧化劑處理之，再以2’, 7’-dichlorofluorescene diacetate(DCFDA)，並測試其活性氧化物之產生。同時亦擬以B3Z 融合瘤細胞分析抗原呈現之表現等。抗氧化藥物對於抗原呈現細胞吞噬凋亡細胞與抗原之影響則將以共軛焦螢光顯微鏡觀察之。在活體實驗方面，擬將以in vivo 方式將含模式抗原之疫苗佐劑，以皮下注射方式打入已經抗氧化劑處理或未處理之小鼠，使產生抗原免疫效應。於數天後將小鼠犠牲，取其脾臟與淋巴結等組織。再分離小鼠之脾臟細胞與淋巴結細胞以進行分析活性氧化物之實驗，測試其T 細胞之增長情況。 同時擬以ELISA 測量細胞上清液proinflammatory cytokines，包括TNFα與 IFN-γ 等之含量，並進行細胞毒殺試驗 (CTL assay)，以瞭解活性氧化物與外在抗原呈現反應間之關係。 同時擬將以real-time RT-TCR 與流式細胞儀分析T 細胞內之chemokine2receptors， granzyme B， perforin 等之表現，並進行chemotaxis assays。此計畫之研究結果將有助於闡明活性氧化物在外生性抗原遞送與cross-presentation 中所扮演之角色。<br> Abstract: Generation of reactive oxygen species (ROS) produced by the innate immune response isimportant in triggering the adaptive immunity. Cross presentation of exogenous antigens resultsfrom processing and presentation of exogenous antigen by the antigen presenting cells with MHC Ito the CD8+ T cells. Here in this research proposal we hypothesize that the production or reactiveoxygen species during antigen processing after immunization plays an essential role for crosspresentation of exogenous antigens via the MHC class I pathway, leading to activation of theeffector function of CD8+ T cells. To examine this hypothesis, the study will be carried out,employing ovalbumin (OVA) as the model antigen, in the murine antigen presenting cells, includingdendritic cells (DCs) and macrophages, isolated from C57BL/6 mice. The APCs will bepre-treated with antioxdants in the presence of adjuvant-induced apoptotic T cells, stimulated withOVA, followed by antigen presentation assay in B3Z hybridoma cells. To correlate with theproduction of ROS, 2’, 7’-dichlorofluorescene diacetate (DCFDA) and hydroethidine (HE) will beused to detect the intracellular respiratory burst activities after treatment with the antioxidants.Effect of antioxidants on the phargocytosis of aoptotic cells and exogenous antigens by the antigenpresenting cells will be examined by a confocal fluorescence microscope.The in vivo experiments will be carried out in the C57BL/6 mice by immunizing theanimals with vaccine adjuvants containing OVA, with or without pretreatment with the antioxidants,and lymphocytes from the secondary lymphoid organs will be isolated two weeks later. Cells willbe analyzed for antigen presentation, antigen-specific T cell proliferation, expression of chemokinereceptors, and CTL activities. The supernatant from the splenocytes will be assayed for theproduction of pro-inflammatory cytokines, including TNF-α and IFN-γ by ELISA. The expressionof chemokine receptors will be analyzed by flow cytometry, and the production of ROS will be2detected by DCFDA and HE. OVA-specific CD8+ T cell cytotoxicc lymphocyte (CTL) assay willbe performed and correlate with the production of ROS after immunization. Studies will beextended by determining if CTL stimulated by ROS is perforin-dependent. Comparison the resultson ROS and CTL assay with the control animals treated with the anitoxidants will elucidate theeffect of antioxidants and the roles of ROS production during cross presentation of exogenousantigens.抗氧化藥物抗原呈現antioxidantsantigen presentation