臺灣博碩士論文加值系統

血管新生在腫瘤生長中扮演關鍵性的角色。有研究指出，寄主的免疫CD4T細胞能監控腫瘤新生血管的生成，然新生血管的免疫感受性究竟如何產生，尚不清楚。本研究的目的即在探討腫瘤血管、CD4T和腫瘤中表現MHCII的浸潤細胞三者之間的關係。先前本實驗室以台灣鼻咽癌EB病毒致癌基因N-LMP1建立了一個以移植方式傳遞的小鼠腫瘤模式，並發現N-LMP1具免疫原性，能引發寄主CD4T細胞反應抑制腫瘤早期的血管生成及生長。為進一步瞭解腫瘤血管的免疫調控，我們假設腫瘤中部分血管新生過程有MHCII細胞的參與，而此過程可受到CD4T細胞的調控。為驗證此假設，我們在經N-LMP1免疫化與非免疫化的兩種小鼠，分別植入腫瘤小塊，在七天後以含有india ink的gelatin經血管灌流標定腫瘤中的血管，再針對腫瘤冷凍切片進行MHCII的螢光染色。之後，再將組織白光及螢光影像以電腦軟體進行MHCII正負血管腔的定量分析。結果發現免疫化組在腫瘤總面積、腫瘤新生面積、總血管數、總血管面積都受到抑制。其中非免疫化組中MHCII+血管佔了總血管數的七成，且此血管在免疫化後被大量抑制，而MHCII-血管則不受影響。為進一步證明MHCII+細胞與血管新生的關聯，我們從腫瘤中純化或去除此細胞並混入matrigel在免疫化或非免疫化的小鼠進行血管生成的觀察。由活體超音波影像與組織切片的結果得知，MHCII+的細胞的確參與了腫瘤最初的血管新生，並且為免疫抑制的目標。我們接著從腫瘤切片中把表現MHCII的血管依管徑進行分類，結果發現以小靜脈(21~30µm)和小動脈 (11~21µm)兩種受免疫化抑制的程度最高。最後以流式細胞儀技術測得CD31-MHCII+和CD31+MHCIIhigh細胞在免疫化後有很明顯細胞性狀的改變。綜合以上的結果可以發現，腫瘤最初的新生血管多以MHCII+細胞參與的機制有關，然此過程可受到寄主CD4T的免疫監控。因此，未來可以腫瘤中表現MHCII的細胞為標的進行腫瘤新生血管的生物標記及疫苗方面的研發工作。

Neovascularization is essential for tumor progression. Although tumor neovessels are known to be under host CD4 T cell surveillance, what makes the neovessels susceptible to CD4 T immunity remains unclear. The aim of this study is to understand the relationship among tumor blood vessels, CD4 T cells and MHCII-expressing tumor infiltrates. Previously, we have established a transplantable tumor model driven by EBV oncogene N-LMP1 in the immunocompetent mice. In the model system, we found that N-LMP1 is immunogenic, capable to induce CD4 T cell immunity to inhibit tumor early neovessel formation. To further understand the CD4 T-mediated regulation of tumor neovascularization, we hypothesized that there are MHCII-expressing cells involved in the neovessel formation, causing the susceptibility of the process for CD4 T immunosurveillance. To test the hypothesis, mice were vaccinated with N-LMP1 to induce CD4 T response (immune group) on day (-4). Mice receiving PBS were used as non-immune control group. Both groups of mice were challenged with tumor fragment on day 0. Seven days later, tumor vessels were labeled in vivo by perfusion of gelatin pre-mixed with india ink. Frozen tumor tissue sections were then immunostain for MHCII. Afterwards, images taken from the white field and fluorescent channel were quantified by computer software Image J. Total tumor area, new tumor area, vessel count and vessel total area were all inhibited in the immune group. While 70% of the total vessels were MHCII+ in the non-immune group, the number and area of MHCII+ vessels were greatly inhibited after immunization. In the meantime, MHCII- vessels were found unaffected. To further study the relationship among MHCII+ cells, neovascularization andCD4 T cells, we sorted or depleted MHCII+ cell population from monosuspended tumor tissue cells, mixed the cells with matigel to test the neovessel formation in the immune and control mice. The results from ultrasound and tissue section demonstrated that MHCII+ cells induced blood vessel formation, and the process of which was inhibited by immunization. Afterwards, the MHCII+ vessels from tumor sections were categorized according to vessel diameter to further analyze the immune target vessels. Veinlet (21~30µm diameters) and arteriole (11~21µm diameters) were found most affected by immunization. Finally, by using flow cytometry, the major tumor tissue cell alteration in immune group was found within CD31-MHCII+ and CD31+MHCIIhigh cell populations, suggesting that both the MHCII-expressing endothelial and microenvironmental cells are targeted by CD4 T cells. Taken together, this study highlights the critical role of MHCII+ tumor infiltrates in the early tumor neovascularization, which makes neovessels susceptible for CD4 T cell surveillance. Vascular MHCII expression may therefore be used as a biomarker for immune-sensitive tumor neovascularization. In addition, MHCII-expressing tumor infiltrating cells may serve as targets for future tumor vaccine development.

目錄
指導教授推薦書............................................................
口試委員會審定書..........................................................
國家圖書館碩士論文電子檔案上網授權書..........................................iii
長庚大學碩士論文著作授權書..................................................iv
誌謝-----------------------------------------------v
中文摘要--------------------------------------------vi
英文摘要--------------------------------------------viii
目錄-----------------------------------------------x
序論-----------------------------------------------1
1.腫瘤與血管-----------------------------------------1
2.血管新生(Angiogenesis)與脈管新生(Vasculogenesis)----1
3.血管新生開關（angiogenic switch）與腫瘤的惡性發展------2
4.腫瘤浸潤細胞與血管生成的關聯--------------------------2
5.CD4T細胞與腫瘤的關聯--------------------------------4
6.CD4 T細胞對血管新生與腫瘤生長的調控機制----------------4
7.鼻咽癌與EB病毒蛋白LMP1------------------------------5
8.N-LMP1腫瘤動物模式及前人研究-------------------------6
9.本研究之假說---------------------------------------8
材料與實驗方法---------------------------------------9
結果-----------------------------------------------15
1.比較非免疫化與免疫化小鼠的腫瘤生長與新生血管的差異--------15
1.1.比較腫瘤總面積、最初移入腫塊面積、腫瘤新生區域面-------15
1.2.比較非免疫化與免疫化腫瘤新生血管之差異---------------16
2.鑑定腫瘤血管的管壁或周遭是否有表現MHCII細胞的存在-------17
3.純化腫瘤中表現MHCII的細胞，鑑定其引發新生血管生成的能力--18
4.鑑定表現MHCII的細胞如何參與腫瘤的血管生成的機制與其受免疫化影響後血管結構上的差異----21
5.非免疫化與免疫化腫瘤中MHCII+與MHCII-血管之管徑比較-----22
6.特定區間的MHCII+血管在非免疫化腫瘤中表現位置-----------23
7.免疫化對MHCII+細胞浸潤的影響------------------------25
8.鑑定腫瘤受免疫化影響後細胞分群之變化-------------------26
8.1. MHCII+CD31+細胞的變化--------------------------26
討論-----------------------------------------------29
參考文獻--------------------------------------------35
附錄-----------------------------------------------40

圖表目次
Fig. 1腫瘤在非免疫化與免疫化的宿主中生長與血管發展的量化比較--------40
Fig. 2非免疫化與免疫化腫瘤血管量化分析--------------------------42
Fig. 3非免疫化與免疫化之腫瘤MHCII螢光染色結果-------------------43
Fig. 4表現MHCII細胞在matrigel中形成血管的能力------------------46
Fig. 5非免疫化小鼠matrigel照射超音波影像與組織免疫染色分析--------48
Fig. 6免疫化小鼠matrigel照射超音波影像與組織免疫染色分析----------49
Fig. 7以Micro-CT影像系統與螢光標定方式觀察matrigel中血管生成情形--50
Fig. 8非免疫化與免疫化腫瘤中MHCII+與MHCII-血管之比較------------52
Fig. 9特定區間血管在非免疫化腫瘤中表現之位置---------------------53
Fig. 10非免疫與免疫化腫瘤中表現MHCII細胞之分析------------------55
Fig. 11非免疫與免疫化腫瘤中CD31相關細胞之分析-------------------57

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