抗血管新生為新興的癌症治療方式。近年來的研究逐漸瞭解免疫系統參與了腫瘤血管新生的調控。因此，瞭解各類腫瘤微環境中免疫因子的變化機制，有助於針對腫瘤的特性研發標靶治療。在諸多免疫因子中，浸潤在腫瘤組織中的巨噬細胞 (TAM)，由於會因腫瘤組織的微環境分化成促進血管新生的角色，已成為當今研究標靶治療的有利目標。鼻咽癌為中國南方與台灣獨特的癌症，其成因與EB病毒的重要致癌基因Lantent membrane protein-1 (N-LMP1)有密切關係。為瞭解在活體中N-LMP1致癌的機轉，本實驗利用過去建立的N-LMP1腫瘤動物模式探討腫瘤發展的機轉。研究分為兩大部分，第一部分為利用RT-PCR方式，去瞭解N-LMP1腫瘤在不同時期(第7、14、21、28天)的腫瘤微環境部分免疫因子表現的情形。第二部分為建立出可模擬TAM參與血管新生的體外試驗。由第一部分結果可知，浸潤在N-LMP1腫瘤中的白血球，以TAM為大宗，CD4-T細胞非常少，但有趣的是，NK細胞也有相當的數量。以細胞激素來看，具促進發炎和血管新生的IL-1β穩定表現，亦有少量IL-10的存在。在趨化素部份，CCL2/CCR2、CCL3/CCR1,CCR5及IP10均存在於腫瘤中，顯示N-LMP1致癌過程對趨化系統的依賴。在血管相關的因子中，CD31及VEGF穩定存在，而其接受者以VEGFR2較穩定。Ang1/Tie2均以第21和28天出現較多。經與小鼠免疫化之後不具生長力的腫瘤比較，發現明顯改變的因子為CD4、NK及IL12的增加，CD31的減少，顯示透過增強免疫的確可經細胞浸潤類型及活性的改變而降低血管新生並抑制腫瘤生長。第二部分以人類單核球細胞株THP-1做測試。我們測試出體外隧道試驗的條件，包括matrigel體積為15μl而凝固時間為1小時，試驗培養條件為20小時。接著以腫瘤內TAM做體外隧道試驗，發現額外添加中和CCL3的抗體後，TAM的通過能力降低，故可初步推斷CCL3具有活化TAM去進行基質分解的能力。本實驗顯示N-LMP1腫瘤的血管新生微環境可經CD4T細胞等細胞免疫機制調控，並且所建立的體外隧道試驗，可用於未來探討TAM促進血管形成過程的機轉。根據初步的結果發現CCL3能夠調節巨噬細胞分解基質的能力，提供未來CCL3可能成為一個抗癌標靶目標的可能性。

Nasopharyngeal carcinoma (NPC) is an unique cancer endemic in South China and Taiwan. And the Epstein-Barr virus-encoded oncogene latent membrane protein- 1 (N-LMP1) is considered as the major promoter in NPC development. To understand the pathogenesis of LMP1, we have established an N-LMP1 tumor mouse model. In the model, this study is focused to analyse the gene expression of various immune and angiogenic factors along tumor progression by RT-PCR. In addition, we developed an in vitro tunnel assay to mimic the angiogenic property of TAM, making tunnels in stroma during tumor angiogenesis. In the RT-PCR study, we found both CD11b and F4/80 (markers of TAM) are consistency expressed, indicating it may be the major population of the infiltrated leukocytes. CD4 is rare, but NK detected by NK1.1 is relatively at higher expression level. IL-1β is stably expressed and IL-10 is rare. CCL2/CCR2, CCL3/CCR1, CCR5 and IP10 all exit in N-LMP1 tumor. We also found stable expression of CD31 and VEGF/VEGFR2. The expression of Ang1/Tie2 is more evident on day 21 and 28. Compare to the tumor becoming non angiogenic in the immunized mice, the apparent differences are the increase of CD4, NK and IL12, and the decrease of CD31. The data suggested that N-LMP1 tumor angiogenesis and progression is complex process heavily involved with various immune cells and factors. In the tunnel assay, we have developed optimal conditions for in vitro tunnel assay and used TAM for a preliminary test. We found that TAM alone could penetrate the matrigel, and the addition of neutratizing anti-CCL3 antibody would greatly reduce the ability. Therefore, CCL3 may be involved in the control of TAM to degrade matrix. The regulatory mechanism through chemokine system warrants further investigation.

論文指導教授推薦書
論文口試委員審定書
長庚大學博碩士紙本論文著作授權書
誌謝
中文摘要
英文摘要
引言……………………………………………………………………1
材料與方法……………………………………………………………14
結果……………………………………………………………………18
討論……………………………………………………………………25
圖表……………………………………………………………………32
附錄……………………………………………………………………60
參考文獻………………………………………………………………61

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