臺灣博碩士論文加值系統

調節性T細胞(regulatory T cells; Treg)，是一群抑制免疫反應的細胞，在免疫病理、器官移植耐受性、阻止自身免疫反應和維持機體免疫平衡中扮演著重要的角色，此細胞為CD4+ T 細胞的亞群，在具有幾個重要的膜表面分子，包含CD25及CD152[又稱毒殺性T細胞第四抗原(Cytotoxic T-Lymphocyte Antigen 4; CTLA-4) ]，以及對於分化成此細胞重要的關鍵轉錄因子，FOXP3(Forkhead box transcription factor)，近年來研究顯示調節性T細胞分化受到表觀遺傳的機制作用(epigenetic mechanisms)，其FOXP3結構中第二個非編碼保守區(conserved non-coding sequences 2; CNS2) 位置，在穩定表現FOXP3之調節性T細胞有高度的去甲基化(demethylation)，所以又稱為Treg cell–specific demethylated region (TSDR)，是目前認定用於調節性T細胞最有價值的標記分子。
大腸直腸癌(colorectal cancer)是目前最普遍發生的惡性腫瘤，而此疾病所造成的死亡率也居高不下，先前文獻發現在此大腸直腸癌組織中聚集了許多調節性T細胞，但卻不清楚此細胞所造成的免疫影響為何。另外，文獻指出，可溶型毒殺性T細胞第四抗原(sCTLA-4)，在自體免疫疾病患者有增加的趨勢，且此分子會與CD80/86分子結合，將干擾APC，而產生阻斷的負調控訊息，而也可能導致疾病的病程不同，更需要進一步研究。本研究目的為探討調節性T細胞在不同疾病中所扮演的角色及其與疾病進程間的關係，與進一步探討調節性T細胞與sCTLA-4間的作用機轉。

材料與方法
　　本實驗經「台中榮民總醫院人體試驗委員會」審查，並具有通過證書，並依照「台中榮總嘉義分院病理檢驗科研究用病理檢體申請審查及審查辦法」將剩餘檢體執行去連結及編碼。
　　本實驗收集之去連結臨床剩餘之胸水檢體共87例，先以蛋白質定量法(protein assay)檢測總蛋白含量 ，並以酵素免疫分析法(ELISA)檢測sCTLA-4、IL-6之含量。另一部分，則收集13例大腸直腸癌蠟塊及3例胃組織，利用滑動式切片機，取下10 m大小之組織與培養細胞株，抽取其genomic DNA進行CNS2區域甲基化定量分析，此部分實驗包含重亞硫酸鹽(bisulfite) 處理、PCR、體外轉錄反應(in vitro transcription) 、鹼基特異性裂解(Base-specific RNA cleavage)及質譜分析。

實驗結果
　　胸腔積液中IL-6表現皆大於300 pg/ml，且總蛋白的含量與sCTLA-4無相關性，而不同癌症其sCTLA-4表現並無差異性，但與先前文獻相比，本偵測濃度高於其他文獻許多，推測在這些癌症中所存在的sCTLA-4具有其功能性，而在肺部疾病患者中發炎性肺疾與肺癌中sCTLA-4表現程度具有顯著性差異。
　　甲基化分析則發現，從胸水中建立的調節性T細胞融合瘤，其甲基化程度只有47%，與其他非調節性T細胞相比明顯低於許多，而檢體中，發現胃幽門螺旋桿菌當中其甲基化比例與調節T細胞融合瘤的比例類似，與先前研究假說有相同之處。
結論
　　調節性T細胞其分泌之sCTLA-4在癌症病患中，並無顯著差異，但在發炎的病患表現卻高於癌症病患，且具有顯著差異，而偵測發炎組織與癌症組織中甲基化的情況，也發現發炎組織的甲基化程度較低，推測發炎環境的組織中或胸腔環境中，發炎介質驅使調節T細胞前往，但是發炎介質對於調節性T細胞具有極大的破壞程度，無法將發炎情況無法降低，且造成調節性T細胞死亡，而在發炎環境中，都可偵測到高表現的調節性T細胞，但是無法確認胸水中細胞的存活性狀況及分布的細胞，需進一步研究利用體外模式確認。而癌症病患中，雖然sCTLA-4表現並無差異，但其表現程度與先前研究偵測濃度相差千倍，推測代表在癌症中調節性T細胞可能具有某種重要角色，而sCTLA-4抑制免疫功能作用也需進一步利用體外模式確認，而目前利用此分子特性做為癌症治療的完全人類單株抗體藥物具有相當大的潛力，而本研究中所建立之調節性T細胞融合瘤在未來體外研究及治療模式上，具有相當大的潛力性。

Regulatory T cell (Treg)-mediated suppression serves as a general mechanism of negative regulation of inflammation and autoinflammatory disorders, allergy, acute and chronic infections, cancers and metabolic inflammation. The Forkhead box transcription factor (FOXP3) is critically involved in Treg development and responsible for determining the suppressive function. Treg cell–specific demethylated region (TSDR) has been identified on the second conserved non-coding sequences 2 (CNS2) in FOXP3. On the other hand, CTLA-4 also provides a negative signal to T cells and thus limiting immune responses. Previously it has been shown that induction and stabilization of FOXP3 expression are under epigenetic control. As cancers usually result from a suppression of specific anti-tumor immune response, we elucidate the possible effect of Treg on local immune function in colorectal cancer and to investigate the possible Treg function in pleural exudate.
With an IRB-approved protocol, we collected 87 samples of pleural effusion, 16 samples of FFPE tissues, and 3 of monoclonal cell lines. Pleural fluid sCTLA levels were determined by ELISA. And based on Treg-specific DNA demethylation within the FOXP3 locus, we quantified of FOXP3 TSDR methylation proportion.
Results show the presence at significantly elevated levels of sCTLA-4 in pneumonia, chronic obstructive pulmonary disease and lung cancer.The cancer with poor overall survival, but the levels of sCTLA-4 is no significant difference between cancers. The ongoing studies will be performed to further clarify the relationship between sCTLA-4 and Treg activity. Epigenetically determined Tregs frequencies could not correlate with CRC in the present study. However, we showed the inflammation disease with high frequencies of FOXP3 demethylation (Tregs) and a very low methylation frequency in Treg hybridoma. Treg hybridoma cells may thus be investigated further as a novel in vitro model.

表目錄 I
圖目錄 II
誌謝 III
中文摘要 IV
英文摘要 8
第一章 概論 9
一、調節性T細胞（Regulatory T cell；Treg） 9
1.1表觀遺傳學（epigenetics）調控機制 9
1.2細胞重要特徵 11
1.3分化機制 15
1.4抑制性機制 17
1.5在臨床上的應用價值 18
1.6臨床治療現況 19
1.7調節T細胞與癌症間關係 19
二、胸腔積液（Pleural effusion）的形成 21
2.1胸腔積液概述 21
2.2成因與分類 21
2.3惡性胸腔積液的臨床意義 24
三、臨床惡性腫瘤的檢查 25
3.1惡性腫瘤與良性腫瘤的特徵 25
3.2臨床檢查 26
第二章 研究動機 30
第三章 材料與方法 32
一、實驗材料 32
二、實驗方法 33
三、實驗流程 43
第四章 結果 44
一、樣本基本資料 44
1.1 胸水積液檢體 44
1.2石蠟組織檢體 47
二、實驗結果 49
2.1胸水積液分析結果 49
2.2蠟塊組織與細胞FOXP3甲基化定量結果 53
第五章、討論 56
1.1探討胸水積液中蛋白表現情形與調節T細胞之表現 56
1.2探討大腸直腸癌組織表現情形與調節T細胞之表現 58
第六章 參考文獻 61
第七章 附件 81
附件1.人體試驗研究計畫許可書 81
附件2 .去連結編碼方法 82
附件3.豐技生物科技有限公司sequenom證書 84

Sakaguchi S, Sakaguchi N, Asano M, Itoh M, &; Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155(3):1151-1164.
2. Josefowicz SZ &; Rudensky A (2009) Control of regulatory T cell lineage commitment and maintenance. Immunity 30(5):616-625.
3. Khavari DA, Sen GL, &; Rinn JL (2010) DNA methylation and epigenetic control of cellular differentiation. Cell Cycle 9(19):3880-3883.
4. Zhou Y, Kim J, Yuan X, &; Braun T (2011) Epigenetic modifications of stem cells: a paradigm for the control of cardiac progenitor cells. Circulation research 109(9):1067-1081.
5. Adeegbe D, Matsutani T, Yang J, Altman NH, &; Malek TR (2010) CD4(+) CD25(+) Foxp3(+) T regulatory cells with limited TCR diversity in control of autoimmunity. J Immunol 184(1):56-66.
6. Taniguchi T, et al. (1983) Structure and expression of a cloned cDNA for human interleukin-2. Nature 302(5906):305-310.
7. Lohr J, Knoechel B, Kahn EC, &; Abbas AK (2004) Role of B7 in T cell tolerance. J Immunol 173(8):5028-5035.
8. Semple K, et al. (2011) Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling. Blood 117(11):3096-3103.
9. Kuczma M, et al. (2009) TCR repertoire and Foxp3 expression define functionally distinct subsets of CD4+ regulatory T cells. J Immunol 183(5):3118-3129.
10. Finger EB &; Bluestone JA (2002) When ligand becomes receptor--tolerance via B7 signaling on DCs. Nature immunology 3(11):1056-1057.
11. Fontenot JD, Gavin MA, &; Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nature immunology 4(4):330-336.
12. Oaks MK, et al. (2000) A native soluble form of CTLA-4. Cellular immunology 201(2):144-153.
13. Zheng Y, et al. (2010) Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 463(7282):808-812.
14. Maruyama T, Konkel JE, Zamarron BF, &; Chen W (2011) The molecular mechanisms of Foxp3 gene regulation. Seminars in immunology 23(6):418-423.
15. Klein L &; Jovanovic K (2011) Regulatory T cell lineage commitment in the thymus. Seminars in immunology 23(6):401-409.
16. Toker A &; Huehn J (2011) To be or not to be a Treg cell: lineage decisions controlled by epigenetic mechanisms. Science signaling 4(158):pe4.
17. Sakaguchi S, Miyara M, Costantino CM, &; Hafler DA (2010) FOXP3+ regulatory T cells in the human immune system. Nature reviews. Immunology 10(7):490-500.
18. Pontoux C, Banz A, &; Papiernik M (2002) Natural CD4 CD25(+) regulatory T cells control the burst of superantigen-induced cytokine production: the role of IL-10. International immunology 14(2):233-239.
19. Somerset DA, Zheng Y, Kilby MD, Sansom DM, &; Drayson MT (2004) Normal human pregnancy is associated with an elevation in the immune suppressive CD25+ CD4+ regulatory T-cell subset. Immunology 112(1):38-43.
20. Magistrelli G, et al. (1999) A soluble form of CTLA-4 generated by alternative splicing is expressed by nonstimulated human T cells. European journal of immunology 29(11):3596-3602.
21. Wolchok JD, et al. (2013) Nivolumab plus Ipilimumab in Advanced Melanoma. The New England journal of medicine.
22. Ha TY (2009) The role of regulatory T cells in cancer. Immune network 9(6):209-235.
23. Ried M &; Hofmann HS (2013) The treatment of pleural carcinosis with malignant pleural effusion. Deutsches Arzteblatt international 110(18):313-318.
24. Gorgun D, Secik F, Midilli K, Akkaya V, &; Yildiz P (2013) Diagnostic and prognostic significance of survivin levels in malignant pleural effusion. Respiratory medicine.
25. Thomas R &; Lee YC (2013) Causes and management of common benign pleural effusions. Thoracic surgery clinics 23(1):25-42, v-vi.
26. Panarelli NC, Schreiner AM, Brandt SM, Shepherd NA, &; Yantiss RK (2013) Histologic Features and Cytologic Techniques That Aid Pathologic Stage Assessment of Colonic Adenocarcinoma. The American journal of surgical pathology.
27. Xiao YF, et al. (2013) microRNA detection in feces, sputum, pleural effusion and urine: Novel tools for cancer screening (Review). Oncology reports 30(2):535-544.
28. Ehrich M, et al. (2005) Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America 102(44):15785-15790.
29. Stockis J, et al. (2009) Comparison of stable human Treg and Th clones by transcriptional profiling. European journal of immunology 39(3):869-882.
30. Ilhan F. BA, Balci TA, Tuten Ozdemir D (2012) Serum and pleural fluid soluble CTLA-4 levels are increased in lung cancer patients Novel Science International Journal of Medical Science 1(5):145-147
31. Erfani N, Razmkhah M, &; Ghaderi A (2010) Circulating soluble CTLA4 (sCTLA4) is elevated in patients with breast cancer. Cancer investigation 28(8):828-832.
32. Yokoyama A, et al. (1992) Interleukin 6 activity in pleural effusion. Its diagnostic value and thrombopoietic activity. Chest 102(4):1055-1059.
33. Sinicrope FA, et al. (2009) Intraepithelial effector (CD3+)/regulatory (FoxP3+) T-cell ratio predicts a clinical outcome of human colon carcinoma. Gastroenterology 137(4):1270-1279.
34. Svensson H, et al. (2012) Accumulation of CCR4(+)CTLA-4 FOXP3(+)CD25(hi) regulatory T cells in colon adenocarcinomas correlate to reduced activation of conventional T cells. PloS one 7(2):e30695.
35. Liu J, et al. (2010) T regulatory cells in cord blood--FOXP3 demethylation as reliable quantitative marker. PloS one 5(10):e13267.
36. Wieczorek G, et al. (2009) Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue. Cancer research 69(2):599-608.
37. Floess S, et al. (2007) Epigenetic control of the foxp3 locus in regulatory T cells. PLoS biology 5(2):e38.
38. Atherton JC &; Blaser MJ (2009) Coadaptation of Helicobacter pylori and humans: ancient history, modern implications. The Journal of clinical investigation 119(9):2475-2487.
39. Lundgren A, Suri-Payer E, Enarsson K, Svennerholm AM, &; Lundin BS (2003) Helicobacter pylori-specific CD4+ CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals. Infection and immunity 71(4):1755-1762.
40. Robinson K, et al. (2008) Helicobacter pylori-induced peptic ulcer disease is associated with inadequate regulatory T cell responses. Gut 57(10):1375-1385.