臺灣博碩士論文加值系統

在正常生理條件下，表現在T細胞上的細胞程式死亡配體1蛋白（Programmed cell death protein 1, PD-1) 可調控體內的免疫反應。但當T細胞處於腫瘤微環境時，因受到抗原不斷的刺激而過度表現PD-1，進而失去對抗腫瘤細胞的能力，這種現象稱為T細胞耗竭（T cell exhaustion）。在臨床上，PD-1免疫療法被用於恢復T細胞能力以對抗癌症，但其治療費用卻十分高昂。過去的臨床研究發現氫氣治療可以減少T細胞上的PD-1表現；但其中的調控機制並未解碼。因此，本研究的目標即是針對氫氣是如何減少T細胞上PD-1的機制進行探討。根據即時定量聚合酶連鎖反應、西方墨點法和流式細胞儀的分析結果顯示，當T細胞生長在富含氫氣的環境中，PD-1的表現量會明顯減少。而氫氣會通過調控細胞質內鈣離子濃度而降低調控PD-1的重要轉錄因子NFATc1活化的數量。另外，我們發現氫氣也會改變T細胞的粒腺體膜電位，且提升細胞的活性。由我們的研究結果可知氫氣是通過調控T細胞內鈣離子濃度進而減少PD-1的表現，這為氫氣在臨床上的應用提供了更有力的證據。此外藉由新發現的調控路徑，我們合理化氫氣可以恢復T細胞活性的機制，也為癌症免疫療法的發展提供新的標的。

Programmed cell death protein 1 (PD-1) is an inhibitory receptor that regulates immune balance in normal physiologic condition. However, T cells in the tumor microenvironment (TME) was found to express high level of PD-1, resulted in a loss of their ability in eliminating cancer. This phenomenon was called T-cell exhaustion. Reversing T-cell exhaustion thus became an inspiring strategy for cancer treatment. PD-1 immunotherapy has been executed for T cell restoration. However the medical cost was too high to be affordable. Previous clinical study has observed the reduced expression of PD-1 on T cells via hydrogen therapy; however, the mechanism underlying remain unclear. We herein aimed to reveal the mechanism on how hydrogen reduce the expression of PD-1 in T cells. We found that hydrogen down-regulated the expression of PD-1 by incubating T cells in hydrogen-rich environment, which was identified by real-time PCR, western blot and flow cytometry. Furthermore, our preliminary data showed that hydrogen suppressed the expression of the activated NFATc1, an important transcription factor for PD-1, via regulating the cellular Ca2+ concentration, as evidenced by decreased amount of activated NFATc1 extracted from cytosol and decreased [Ca2+] in cytosol. In addition, we confirmed that hydrogen exerted an effect on the mitochondrial membrane potential. In summary, we discovered that hydrogen-mediated PD-1 downregulation in T cell was manipulated by Ca2+-mediated pathway, and these results rationalize the application of hydrogen in clinical practice. Moreover, as hydrogen-mediated T cell restoration pathway was uncovered, new targets can be discovered and considered to be used in future development of anti-cancer immunotherapy.

誌謝 I
摘要 II
ABSTRACT III
目錄 1
圖目錄 4
表目錄 6
第一章 文獻回顧 7
1.1 緒論 7
1.2 氫氣在醫療上的應用 8
1.2.1 氣體療法 8
1.2.2 氫氣（Hydrogen gas; H2）的特性（針對治療疾病） 8
1.2.3 氫氣應用於臨床治療的方式及效果 10
1.2.4 氫氣由抗發炎到調控機制的進展 12
1.3 程序性細胞死亡因子（PROGRAMMED CELL DEATH-1, PD-1） 13
1.3.1 發現及命名 13
1.3.2 結構組成及特性（參考圖1-2與圖1-3） 13
1.3.3 PD-1和PD-1配體的作用（參考圖1-3） 14
1.4 癌症免疫療法（CANCER IMMUNOTHERAPY） 15
1.4.1 T細胞的免疫作用 15
1.4.2 癌症免疫療法歷史和原理 16
1.4.3 T細胞耗竭 （T cell exhaustion） 18
1.4.4 癌症免疫療法的不足之處 18
1.5 T細胞調控PD-1表現的轉錄因子 19
第二章 實驗假說與項目 22
2.1 實驗假說 22
2.2 實驗項目 23
2.3 實驗設計示意圖 23
第三章 實驗材料與方法 24
3.1 實驗材料 24
3.1.1 藥品 24
3.1.2 試劑 26
3.1.3 抗體 26
3.1.4 引子序列 27
3.1.5 儀器 28
3.1.6 細胞株 30
3.1.7 氣體 30
3.2 實驗方法 31
3.2.1 細胞培養（Cell culture） 31
3.2.1.1 細胞株生長條件 31
3.2.1.2 培養基及緩衝液配製 31
3.2.1.3 細胞繼代 32
3.2.1.4 細胞計數與種細胞 32
3.2.1.5 細胞冷凍與解凍 33
3.2.2 RNA 萃取 （RNA extraction） 34
3.2.3 將mRNA轉成cDNA 35
3.2.4 即時聚合酶連鎖反應 （Real-time PCR） 36
3.2.5 蛋白質萃取 （Protein extraction） 37
3.2.5.1 全蛋白萃取（Total protein extraction） 37
3.2.5.2 核蛋白萃取 37
3.2.5.3 粒腺體蛋白萃取 （全程於冰上進行） 38
3.2.5.4 蛋白質定量 39
3.2.6 粒腺體膜電位分析 40
3.2.7 西方墨點法 （Western Blot） 41
3.2.8 測量細胞質內鈣離子濃度 46
第四章 實驗結果與討論 47
4.1 氫氣不會對細胞的增殖生長造成影響 47
Part I : 氫氣的作用形式 48
Part II：氫氣對細胞存活率的影響 51
4.2 氫氣對EL4細胞中PD-1表現的影響 53
Part I : 氫氣對PD-1 mRNA表現量的影響 53
Part II : 氫氣對PD-1 protein表現量的影響 56
4.3 NFATC1是主要調控PD-1表現的轉錄因子 59
4.4 氫氣通過平衡細胞質內鈣離子濃度調控T細胞內PD-1的表現 63
Part I: 細胞質內鈣離子濃度對PD-1表現的影響 63
Part II: NFATc1蛋白質含量分析 66
4.5 氫氣可以增強粒腺體活性 68
4.6 氫氣可以增強T細胞抗腫瘤能力 71
第五章 結論與未來展望 72
第六章 參考文獻 74

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