賴明宗臺灣大學：免疫學研究所徐大宙Hsu, Ta-ChouTa-ChouHsu2007-11-292018-07-092007-11-292018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/63313Notch訊息在免疫系統裡扮演許多重要的角色。此訊息不僅參與在決定T細胞發育過程中的細胞分化，並可以調節成熟T細胞的活化。在果蠅和哺乳類中的研究指出，內噬作用 (endocytosis)是 Notch訊息傳遞之起始過程所必須。目前對於在 T細胞中，內噬作用是否參與 Notch訊息傳導並不清楚。在內噬作用的過程中，標的蛋白會被單泛素 (monoubquitination)作為內部化的訊息。Deltex是含 RING finger domain的 E3 ligase，在不同細胞內 deltex對於 Notch的調控角色並不相同。在本研究中我們探討 deltex在 T細胞中對 Notch訊息調控所扮演的角色，並分析 deltex是否為 Notch受器內部化所需的 E3 ligase。在過度表現 deltex的 DO11.10細胞，我們發現經由 Delta-like-1 (Dl-1)配體誘發而產生的活化型 Notch intracellular domain (NICD)少於控制組細胞，Notch標的基因 HES-1的表現也同時減少。而下調 deltex的 DO11.10則對 Dl-1誘發的 HES-1表現有增加的作用。研究結果顯示 deltex對 NICD產生及 Notch訊息有負面的調控，並不支持 deltex為活化 Notch所需的 E3 ligase。 在探討內噬作用在 NICD產生的角色上，我們首先以顯性抑制型Dynamin (Dyn K44A)阻撓內噬作用。但在 Jurkat細胞誘發 Dyn K44A表現後，卻造成明顯的細胞凋亡。因此改用顯性抑制型的 Eps15 (Eps15 DIII)阻斷內噬作用的形成，探討在 Dl-1配體刺激下 Notch訊息的傳遞是否受阻。我們意外發現 Eps15 DIII可以大量增加 Dl-1刺激後 NICD的產生。由細胞表面受器染色，我們發現在 Eps15 DIII的 DO11.10細胞上的 Notch並沒有增多，顯示 Eps15 DIII並非透過增加細胞表面 Notch受器的表現而使 NICD增加。但是我們也發現，Eps15 DIII並沒有明顯增加 NICD轉錄活化通的分子 HES-1的表現，顯示 NICD的大量增加與 NICD錄錄活化間有很大差異。 我們結果指出，內噬作用很可能在 Notch受體活化後 NICD的產生上扮演關鍵性角色，但 deltex可能不是 Notch活化時，執行單泛素化的 E3 ligase。然而在內噬作用及 NICD產生及轉碌活化上仍有很多過程不清楚，有待更進一步詳細的探討。Notch signals play critical roles in immune system. Notch signal determine the development of T cell lineage and the differentiation of mature T cell. Recent studies in Drosophila and mammalian suggest that endocytosis is required for the generation of Notch intracellular domain (NICD) and initiation of Notch signal. Whether endocytosis is required for Notch signal in T cell remains unclear. Monoubquitination of target protein provides the internalization signal during endocytosis. Deltex is a Notch-binding E3 ligase containing RING finger. In this study, we investigated the role of endocytosis in the generation of Notch signal in T cell, and examine the possibility whether deltex is the E3 ligase for Notch receptor monoubquitination. We found that overexpression of deltex in DO11.10 T cell led to reduced NICD generation after delta-like-1 (Dl-1) stimulation compared to YFP control. Dl-1 induced HES-1 expression was also decreased in deltex-overexpressiing DO11.10 than YFP control. In contrast, knock-down of deltex slightly increased Dl-1-induced HES-1 expression in DO11.10 T cell related to pLL3.7 control. Our results indicate that deltex may play negative role in regulating Notch signal, and that deltex is likely not the E3 ligase required for Notch signal activation. To investigate the role of endocytosis in NICD generation, we first used dominant negative Dynamin2 (Dynamin2 K44A) to block endocytosis in T cells. The inducible expression of Dyn2 K44A in Jurkat cells led to apoptosis. We therefore switched to dominant negative Eps15 (Eps15 DIII) to study the effect of endocytosis blockage on NICD generation. To our surprise, we found that Eps15 DIII profoundly enhanced NICD generation after Dl-1 stimulation in DO11.10 cells. The cell surface staining of Notch indicates that Eps15 DIII expression did not lead to elevated Notch receptor accumulation on cell membrane for the observed NICD generation. However, the expression of NICD transcription target HES-1 was not significantly increased in Eps15 DII-expressing DO11.10 cells, suggesting there is difference between the generation of NICD and the activation of NICD transcription. Our results indicate that endocytosis is participated in the process of NICD generation after Dl-1 ligand stimulation, yet deltex may not be the E3 ligase that monoubquitinates Notch receptor after Dl-1 ligad stimulation. Further studies are required to reveal the detailed mechanism on how endocytosis is involved in NICD generation and on how NICD transcription activity is activated.目錄 中文摘要..................................................i 英文摘要................................................iii 目錄......................................................v 第一章 緒論.............................................1 1.1內噬作用 (Endocytosis)簡介..............................1 1.1.1泛素 (ubquitin)作為內化訊息 (internalization signal)..2 1.1.2 Epidermal growth factor pathway substrate 15 (Eps15) ..3 1.1.3 Dynamin.............................................4 1.2 Notch..................................................4 1.2.1 Notch 之簡介........................................5 1.2.2 Notch 調節T細胞發育及分化...........................6 1.2.3 Deltex 為Notch 交互作用蛋白..........................6 1.2.4 內噬作用對Notch訊息的調控..........................7 1.3 研究方向與目的........................................8 第二章 材料與方法.........................................10 2.1 細胞株及其培養.......................................10 2.1.1 哺乳類、人類細胞株................................10 2.1.2 細胞培養..........................................10 2.2 試劑.................................................10 2.3 抗體.................................................11 2.4 質體構築.............................................11 2.4.1 Delta-like-1 ......................................11 2.4.2 pGeneB/Dynamin2...................................12 2.4.3 pGeneB/Eps15 DIII..................................12 2.5 質體DNA的轉染.......................................12 2.5.1 Calcium phosphate..................................12 2.5.2 電穿孔法 (electroporation) .......................12 2.5.3 反轉錄病毒感染法..................................13 2.5.4 pGeneSwitch 誘導系統 (inducible system) ..........13 2.6 固著式 notch 配體活化Notch受體......................14 2.7 內噬作用分析 (endocytosis assay) .....................14 2.8 西方墨點法...........................................14 2.9 細胞萃取液的製備.....................................15 2.9.1 全細胞萃取液的製備................................15 2.9.2 細胞核萃取液的製備................................15 2.10 萃取RNA 及反轉錄PCR................................16 2.11 重組蛋白質純化......................................17 第三章 結果...............................................18 3.1 固著式 Delta-like1 配體在 DO11.10中可活化 Notch訊息.18 3.2 在T細胞中 deltex可調降Dl-1活的 Notch訊息...........18 3.3 誘導表現顯性抑制型的 dynamin2 K44A會導致細胞凋亡.....19 3.4 顯性抑制型的 Eps15DIII會導致Notch訊息增強，但並沒有增加Notch在細胞膜上的表現量..............................21 第四章 討論...............................................23 參考文獻.................................................28 圖表.....................................................35 附錄 ....................................................521767644 bytesapplication/pdfen-UST 細胞內噬作用NotchEndocytosisEps15dynamin2otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63313/1/ntu-95-R94449011-1.pdf