臺灣博碩士論文加值系統

中文摘要
EB病毒的致癌基因LMP1已知與許多腫瘤具有高度相關性。在台灣鼻咽癌檢體所分離出的LMP1(稱為NLMP1)，發現與過去在B淋巴癌所分離出的LMP1(稱BLMP1)有部分序列的不同。更有多篇報導指出，NLMP1是一種致癌性強，免疫原性弱的致癌基因。然而在本實驗室過去所建立的小鼠腫瘤模型中顯示，EB病毒所表現的NLMP1致癌蛋白質的確是具有免疫原性的。若以放射線去活性的NLMP1腫瘤單一細胞進行免疫注射，可使80%的小鼠獲得對抗NLMP1腫瘤的免疫能力。此外也發現，此免疫注射所引起的免疫反應與T細胞的活性有所關。沿此，更進一步鑑定出NLMP1的CD8+ T細胞抗原決定區的一個NLMP1抗原性胜肽。近年來的研究逐漸對樹突細胞在NK及T細胞反應的關鍵角色，有所認知，並且藉由樹突細胞呈現抗原，活化對抗腫瘤CD4及CD8 T細胞免疫反應的技術亦漸趨成熟。在本實驗的研究中，我們利用攜帶NLMP1基因的重組腺病毒以及NLMP1抗原性胜肽加諸在成熟樹突細胞，以期進一步瞭解此方式在活化對抗NLMP1腫瘤的專一性免疫反應的效果。結果發現，本實驗室所建構的AdNLMP1重組腺病毒的確可以做為NLMP1的載體，並在細胞中表達NLMP1。在MOI 250時對於成熟樹突細胞的感染率，約有40％。另外，此種重組腺病毒感染樹突細胞後，表現（CD40、CD80、CD86、MHC I和MHC II），並且細胞素IL-6的分泌大量增加。在體外，以重組腺病毒感染的樹突細胞或加入抗原性胜肽的樹突細胞均可以活化具有對NLMP1專一性毒殺能力的殺手T細胞。在活體中，打入抗原性胜肽樹突細胞亦可以活化對抗專一性NLMP1腫瘤的效果。此外，本實驗藉由抗體去除活體內細胞的方式，進一步發現完整的抗NLMP1腫瘤的免疫機制尚與CD4+ T細胞和自然殺手細胞有關。因此，本研究結果，提供了未來利用NLMP1蛋白質進行腫瘤免疫療法的可行性。然而，將來在NLMP1腫瘤疫苗的設計應朝向多邊活化CD4 T，CD8 T及NK反應的方向努力。

Abstract
EBV-encoded oncogene LMP1 has been known to be highly associated with several malignancies. LMP1 isolated from Taiwan’s NPC patient biopsy (NLMP1) is different from the gene originally identified in Burkitt’s lymphoma (BLMP1) by 10 amino acid deletion at C-terminal end and multiple point mutations throughout the gene. In addition, it has been documented that NLMP1 oncoprotien exerts longer half life, higher tumorigenicity and no immunogenicity. However, by an EBV-NLMP1 tumor animal model established in this laboratory, we have demonstrated irradiated NLMP1 tumor cells can induce a strong T cell-mediated anti-tumor immunity. A CD8+ T cell epitope of NLMP1 (Np) has also been identified. Recently, many reports illustrated the interplay among T, NK and dendritic cells (DC). And DC technology in CD4+ or CD8+ T cell activation has also been developed. Here, to test whether NLMP1 itself can be presented by DC to activate CTL response, the system that DC infected with recombinant adenovirus carrying NLMP1 (AdNLMP1) or pulsed with Np was established. Firstly, we showed the expression of AdNLMP1-derived LMP1 protein was transient for several days after infection. Secondly, the infectivity of AdNLMP1 to DC from of BALB/c mice is ~40% at MOI 250. Thirdly, CD40, CD80, CD86, MHC I and MHC II expression on DC was positive after viral infection. Fourthly, the production of IL-6 is also elevated after induction. In subsequent in vitro and ex vivo experiments, AdNLMP1-infected or Np-pulsed DC (AdNLMP1-DC or Np-DC) exhibited the ability to activate anti-NLMP1 tumor CTL. Furthermore, Np-DC immunized mice were challenged with NLMP1 tumor. A significant delayed tumor growth was found. In addition, to understand the immune network underlying the tumor prevention, in vivo antibody depletion experiment upon immunization revealed that CD4+ T and NK cells are also involved in the anti-tumor immunity. Altogether, our results suggest that a NLMP1-based DC vaccine can be designed to achieve a full scale of anti-NLMP1 immunity in vivo consisting CD4+ T, CD8+ T and NK response.

Contents
指導教授推薦書…………………………………………………………
口試委員審定書…………………………………………………………
授權書…………………………………………………………………….
電子檔案上網授權書……………………………………………………..
致謝………………………………………………………………………..
Abstract…………………………………………………………………….
中文摘要…………………………………………………………………..
Introduction……………………………………………………………....1
Material and Methods…………………………………………………….3
Results…………………………………………………………………...11
Discussion…………………………………………………………...…..16
Reference………………………………………………………………..20
Figures…………………………………………………………………..27

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