臺灣博碩士論文加值系統

近年來國人的頭頸癌罹癌率有快速成長的趨勢，且根據目前研究
顯示頭頸癌的病情發展與免疫功能的強弱有著密切關係，其中以T 細
胞參與扮演著重要的角色，當T 細胞維持自體免疫耐受性及抗原辨認
功能受影響時，可能會改變並抑制對腫瘤細胞的免疫反應，而使癌症
逃避免疫系統的攻擊。在本研究中，我們利用流式細胞儀分析頭頸癌
病人周邊血淋巴球中T 細胞及其亞群的分佈情形，探討其與頭頸癌疾
病發展的可能相關性。首先我們發現在頭頸癌病人周邊血中
CD4+CD25+的調節性T 細胞數量較正常健康人高，其中也包括CD4+
CD25High 及CD4+Foxp3+等細胞族群。另一方面，同時發現一群與分泌
發炎激素IL-17 相關的CD4+ T 細胞亞群: Th17 細胞，其比例也有大幅
增加的情形，且增加趨勢與腫瘤發展嚴重程度相關。同時病人周邊血
淋巴球刺激活化後所產生的IL-17 以及和Th17 生成相關細胞激素
IL-21 與TGF-β 皆有大幅上升的情形，顯示這群IL-17+細胞在腫瘤發
展過程中有其影響性。這些免疫細胞分佈的改變，可能與腫瘤發展及
其治療效率或復發機率相關，若能深入了解其中相關的腫瘤免疫反應
的發生及調節機制，有利於未來發展癌症療法，以控制腫瘤惡化及預
防復發。

The role of anti-tumor immunity in relation to head and neck squamous
cell carcinoma (HNSCC) has been intensely studied in the past few years.
It is now becoming clear that the complex interaction between HNSCC
and immune cells plays an important part in determining tumor growth
and progression. It has also led to the identification of a distinct
suppressor cell population known as regulatory T cells (Treg) that play a
crucial role in maintaining T-cell tolerance to self-antigens. Therefore, the
current study aimed to utilize multicolor flow cytometric analysis to study
the frequency and phenotype of Treg cells in peripheral blood
lymphocytes (PBL) of HNSSC patients. We found that compared with
healthy volunteers, the percentages of circulating CD4+CD25+ cells were
increased in HNSCC patients, and this cell subset contained
CD4+CD25high and CD4+CD25+Foxp3+ cells. In addition, the frequency of
CD4+ cells (Th17) was also investigated, and patients with HNSCC had a
higher proportion of IL17+ T cells in peripheral blood. Notably, the
increased prevalence of IL17+ T cells was associated with clinical stage.
Furthermore, the concentrations of IL-17、TGF-β and IL-21 cytokines in
patients’ PBMC culture supernatant were significantly increased, and
evaluated the potential association of Th17 cells with HNSCC. These
results indicate that Treg and IL-17+ cells may contribute to tumor
pathogenesis of HNSCC. Further studies of molecular mechanisms that
are involved in the function of Treg and Th17 with different immune cell
subsets are urgently required to help develop immunotherapeutic
strategies that might overcome tumor immunsuppression.

指導教授推薦書……………………………………………………
口試委員會審定書…………………………………………………
授權書……………………………………………………………… iii
誌謝………………………………………………………………… iv
中文摘要…………………………………………………………… v
英文摘要…………………………………………………………… vi
縮寫表……………………………………………………………… vii
目錄………………………………………………………………… viii
圖目錄……………………………………………………………… ix
附錄目錄…………………………………………………………… xiii

第一章 背景………………………………………………………… 1
第二章 實驗材料與方法……….…………………………………… 11
第三章 研究結果……….…………………………………………… 15
第四章 討論……….………………………………………………… 26
第五章 參考文獻……….…………………………………………… 35
第六章 附圖……….………………………………………………… 42
第七章 附錄……….………………………………………………… 71

圖一. 比較健康受試者與不同腫瘤發展程度之HNSCC 病人周邊血單
核細胞中CD4+與CD8+淋巴細胞的分佈.………………………..42
圖二. 依照腫瘤大小，比較HNSCC 病人周邊血單核細胞中CD4+與
CD8+淋巴細胞的分佈……………………………………………..43
圖三. 依HNSCC 病人發生淋巴結轉移與否，比較其周邊血單核細胞
中CD4+與CD8+淋巴細胞之分佈…………………………………44
圖四. 比較不同腫瘤發生部位的HNSCC 病人之周邊血中CD4+及
CD8+淋巴細胞的分佈……………………………………. ……....45
圖五. 比較不同腫瘤發展時期CD4+CD25+、CD4+CD25high 與CD4+
Foxp3+細胞在HNSCC 病人的周邊血單核細胞中佔有比例……46
圖六. 依照腫瘤大小，比較HNSCC 病人周邊血淋巴細胞中調節性T
細胞比例…………………………. ……………………. ………...47
圖七.依HNSCC 病人發生淋巴結轉移與否，比較其周邊血單核細胞中
調節性T 細胞之分佈……………………. ……………………. ..48
圖八. 比較不同腫瘤生成部位的HNSCC 病人，其調節性T 細胞在周
邊血單核細胞中占有比例……………………. ………………….49
圖九. 比較HNSCC 病人與健康受試者周邊血單核細胞經刺激後
TGF-β1 之生成量……………………. ……………………. …….50
圖十. HNSCC 病人與健康受試者周邊血單核細胞刺激後，發炎相關細
胞激素IL-10 生成量之比較……………………. ……………….51
圖十一. 比較健康受試者與不同腫瘤發展程度之HNSCC 病人周邊血
單核細胞中CD4+IL-17+與CD8+IL-17+比例…………………….52
圖十二. 依照腫瘤大小，比較IL-17+ T 細胞在HNSCC 病人周邊血淋
巴球中所占之比例……………………. ……………………. …...53
圖十三.依HNSCC 病人發生淋巴結轉移與否，比較其周邊血單核細胞
中IL-17+ 細胞之分佈……………………. ……………………...54
圖十四. 比較不同腫瘤生成部位的HNSCC 病人，其IL-17+細胞在周
邊血單核細胞中分佈……………………. ………………………55
圖十五.比較HNSCC 病人與健康受試者周邊血單核細胞經刺激後
IL-17 生之生成量……………………. ……………………. ……56
圖十六. HNSCC 病人與健康受試者周邊血單核細胞經刺激後，比較其
IL-21 生成量……………………. ……………………. …………57
圖十七. HNSCC 病人與健康受試者周邊血單核細胞經刺激後，比較其
發炎相關細胞激素IL-1β 之生成量……………………. ………58
圖十八. HNSCC 病人與健康受試者周邊血單核細胞經刺激後，其發炎
相關細胞激素IL-6 生成量之比較………………………………59
圖十九. 利用CD4+CD25+、CD4+CD25high 與CD4+Foxp3+細胞之間的
相關性，比較調節性T 細胞在不同腫瘤發展時期的分佈………60
圖二十. 利用CD4+ CD25+、CD4+CD25high、CD4+ Foxp3+ 與CD4+
IL-17+細胞之間的相關性，比較調節性T 細胞與Th17 在不同腫
瘤發展時期的分佈……………………. ……………………. …..61
圖二十一.健康受試者與不同腫瘤發展程度之HNSCC 病人周邊血單
核細胞中CD4+ IL17+ Foxp3+、CD4+ IL17+ Foxp3-與CD4+ IL17-
Foxp3+細胞分佈之比較……………………. ……………………62
圖二十二. 依照腫瘤大小區分，比較HNSCC 病人周邊血單核細胞中
CD4+Foxp3+IL17+、CD4+Foxp3+IL17-與CD4+Foxp3-IL17+細胞比
例……………………. ……………………. ……………………...63
圖二十三.依HNSCC 病人發生淋巴結轉移與否，比較其周邊血單核細
胞中CD4+Foxp3+IL17+、CD4+Foxp3+IL17-與CD4+Foxp3-IL17+細
胞之分佈……………………. ……………………. ……………...64
圖二十四. 比較不同腫瘤生成部位的HNSCC 病人，其周邊血單核細
胞中CD4+ Foxp3+IL17+、CD4+ Foxp3+ IL17-與CD4+ Foxp3-IL17+
細胞比例……………………. ……………………. ……………...65
圖二十五. 比較健康受試者與不同腫瘤發展程度之HNSCC 病人周邊
血單核細胞中，其CD4 細胞的Foxp3 平均螢光強度表現……….66
圖二十六. 比較不同腫瘤發展時期HNSCC 病人周邊血單核細胞中，
其CD8+CD25+、CD8+ Foxp3+與CD8+CD25+Foxp3+細胞之比
例……………………. ……………………. ……………………...67
圖二十七. 依照腫瘤大小，比較HNSCC 病人周邊血單核細胞中其
CD8+CD25+、CD8+ Foxp3+與CD8+CD25+Foxp3+細胞之比例…68
圖二十八. 依HNSCC病人發生淋巴結轉移與否，比較其CD8+CD25+、
CD8+ Foxp3+與CD8+CD25+Foxp3+細胞在周邊血單核細胞中之比
例……………………. ……………………. ……………...............69
圖二十九. 比較不同腫瘤生成部位的HNSCC 病人，其CD8+調節性T
細胞在周邊血單核細胞中占有比例……………………. ……….70

附錄一、ELISA 試劑配方……………………. ……………………….71
附錄二、Clinicopathologic characteristics of patients with HNSCC who
donated PBMC for this study……………………. ………………..72
附錄三、Classification of HNSCC patients number by clinicopathologic
characterization……………………. ……………………. ……….73
附錄四、Univarite analysis of clinicopathologic characterization for
IL-17+ cell expression in 131 HNSCC patients…………………....74
附錄五、Univarite analysis of clinicopathologic characterization for
regulatory T cell expression in 131 HNSCC patients……………...75
附錄六、Univarite analysis of clinicopathologic characterization for
CD8 regulatory T cell expression in 131 HNSCC patients………..76
附錄七、比較健康受試者與HNSCC 病人周邊血單核細胞中IFN-γ+細
胞……………………. ……………………. ……………………..77
附錄八、 HNSCC 病人與健康受試者周邊血單核細胞經刺激後，其發
炎相關細胞激素TNF 之生成量，並依照不同腫瘤發展時期區
分……………………. ……………………. ………………….…78

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