臺灣博碩士論文加值系統

癌症已經成為現代化社會中主要死因之一，不僅位居台灣十大死因之首位，美國癌症學會的資料也顯示，在2018年全球新增1810萬的癌症病例，且有960萬人死於癌症，世界衛生組織（WHO）推估2023年之後，全球每年將有2,200萬新增的癌症病例。在癌症免疫治療標的的研究中，除了標靶單株抗體的發展，吲哚胺2,3-雙加氧酶（indoleamine 2,3-dioxygenase, IDO）與色氨酸2,3-雙加氧酶（tryptophan 2,3-dioxygenase, TDO）的小分子抑制劑近年來也被認為是一個新穎的癌症免疫治療藥物。IDO、TDO這兩個酵素均參與在代謝色氨酸（tryptophan）的過程，是犬尿氨酸（kynurenine）途徑的速率決定步驟（rate-limiting step）之限速酶。色氨酸代謝為犬尿氨酸後可以降低T細胞的活性以及增殖能力，也會使得樹突細胞（dendritic cell）將CD4+ T細胞轉化為調節型T細胞（T regulatory cells, Tregs）。這兩種調控作用均會影響並使得宿主的免疫功能受到抑制，在腫瘤的微環境（microenvironment）中幫助腫瘤細胞從免疫監控中逃脫，因而增加腫瘤細胞的免疫耐受性；因此本篇研究目的為建立細胞內IDO/TDO活性測定平台，以及篩選新穎的IDO/TDO抑制劑。在本篇研究中分別透過HT-29以及U-87MG細胞株表達IDO和TDO兩種蛋白，再給予色胺酸及藥物反應，收取培養細胞之上清液，使用液相層析質譜儀（LC-MS）分析上清液中之色胺酸和犬尿胺酸之含量，評斷藥物是否有抑制IDO或TDO酵素活性之功能。在本篇實驗結果顯示，由禾伸堂生技公司所提供的化合物AB-8198及其經由許豪仁老師實驗室修飾後之衍生物在細胞平台內皆沒有抑制IDO酵素活性之能力；而AB-8198和其衍生物HS14705、HS14708在細胞平台中有能力抑制TDO酵素活性，但修飾後之衍生物在細胞平台中可能受到細胞膜等因素干擾，無法順利進入細胞內，造成半抑制濃度未能優於AB-8198。

Cancer has become one of the leading causes of death in modern society. Not only is it the top ten cause of death in Taiwan, but the American Cancer Society also shows that in 2018, 18.1 million cancer cases were added globally, and 9.6 million people died of cancer. The World Health Organization (WHO) estimates that after 2023, there will be 22 million new cancer cases worldwide each year. In the study of cancer immunotherapy specimens, in addition to the development of targeted monoclonal antibodies, indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) doped with small molecule inhibitors is also considered to be a novel cancer immunotherapy drug. The two enzymes IDO and TDO are involved in the process of metabolizing tryptophan, which is the rate-limiting enzyme of the rate-limiting step of the kynurenine pathway. Metabolism of tryptophan into kynurenine can reduce the activity and proliferation of T cells, and also make dendritic cells convert CD4+ T cells into T regulatory cells (Tregs). Both of these regulatory effects will affect and suppress the immune function of the host, and help tumor cells escape from immune surveillance in the microenvironment of the tumor, thus increasing the immune tolerance of the tumor cells; therefore the purpose of this research In order to establish an intracellular IDO/TDO activity measurement platforms and to screen novel IDO/TDO inhibitors. In this study, two kinds of proteins, IDO and TDO, were expressed through HT-29 and U-87MG cell lines, and then tryptophan and drug were added. The supernatant of the cultured cells was collected and the contents of tryptophan and kynurenine in the supernatant was analyzed by liquid chromatography mass spectrometry (LC-MS) to judge whether the drug has the function of inhibiting the enzyme activity of IDO or TDO. The experimental results show that the compound AB-8198 provided by He Shen Tang Biotechnology Company and its derivatives modified by Dr. Hsu’s laboratory have no ability to inhibit IDO enzyme activity in the cell platform; meanwhile, AB-8198 and its derivatives HS14705, HS14708 have the ability to inhibit TDO enzyme activity in the cell platform, but the modified derivatives may be interfered by cell membranes and other factors in the cell platform, and cannot enter the cell smoothly, resulting in IC50 concentration failed to outperform AB-8198.

摘要 I
ABSTRACT II
目錄 IV
圖表目錄 VI
壹、研究背景 1
一、免疫治療（Immune therapy） 1
1.免疫反應（Immune response） 1
2.免疫治療（Immunotherapy） 2
二、吲哚胺2,3-雙加氧酶（Indoleamine 2,3-dioxygenase, IDO） 4
三、色胺酸2,3-雙加氧酶（Tryptophan 2,3-dioxygenase, TDO） 5
貳、研究動機與目的 6
參、實驗材料與方法 7
一、實驗儀器 7
二、藥品與試劑 8
三、實驗方法 12
1、細胞培養 （Cell culture） 12
2、細胞活性測試（Cell viability assay） 14
3、吲哚胺2,3-雙加氧酶酵素活性半抑制濃度測定 （IDO activity IC50 assay） 14
4、色胺酸2,3-雙加氧酶酵素活性半抑制濃度測定 （TDO activity IC50 assay） 15
5、西方墨點法 （Western blotting） 15
6、西方墨點法量化（Western blotting quantitation） 20
7、HPLC-MS標準品製備與校正曲線 20
8、液相層析質譜儀 (Liquid Chromatography-Mass Spectrometry, LC-MS) 分析條件設定 21
9、實驗數據統計分析（Statistical analysis） 22
肆、實驗結果 23
一、測試化合物是否會對人類內皮細胞（HMEC-1）具有細胞毒性之影響 23
二、利用IFN-刺激人類大腸癌細胞（HT-29&HCT-116）表達IDO 23
三、確認人類星形膠質母細胞瘤（U-87MG）表現TDO 23
四、使用HPLC-MS 分析化合物對吲哚胺2,3-雙加氧酶（IDO）之親和力 24
五、HPLC-MS分析化合物對色胺酸2,3-雙加氧酶（TDO）之親和力 24
伍、討論 37
陸、結論 39
柒、參考文獻 40

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