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研究生:許翰誠
研究生(外文):Han-ChengHsu
論文名稱:含雙色胺酸功能區氧化還原酶在T細胞活化及增生中扮演的調控性角色
論文名稱(外文):The Regulatory Role of WW Domain-Containing Oxidoreductase in T cell Activation and Proliferation
指導教授:徐麗君徐麗君引用關係
指導教授(外文):Li-Jin Hsu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學檢驗生物技術學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:81
中文關鍵詞:含雙色胺酸功能區氧化還原酶T細胞
外文關鍵詞:WW Domain-Containing OxidoreductaseT cell
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T細胞調控的免疫反應對於人體是重要的,藉由對先天以及後天性免疫的調控能消滅惡性細胞的增生或者是抵抗外來病原入侵。而T細胞活化及增生過程中,T cell receptor (TCR)接收到刺激而引發的訊息傳遞是關鍵性的過程。因此任何能調控此訊息傳遞的因素都是值得去探討及研究的。含雙色胺酸功能區氧化還原酶(WW domain-containing oxidoreductase),簡稱WWOX或WOX1,有許多重要的生理意義,包括腫瘤抑制蛋白的角色、調控胚胎發育、神經發育及發展、調節生物體內的代謝作用,而目前發現WWOX對於免疫系統似乎有所調控。近幾年我們實驗室團隊發現Wwox缺少(Wwox-/-)的小鼠,其thymocyte的分化及發育有嚴重的缺失。但是在這樣的小鼠體內,依然能在血液中發現成熟的T細胞,而這些T細胞在缺少Wwox後,是否和含有Wwox (Wwox+/+)的T細胞在接收到刺激後具有同樣的特性,這部分的研究是缺乏的。因此我們探討WWOX在T細胞活化及增生中扮演的角色。我們利用全身缺少Wwox的小鼠以及專一性在T細胞中缺少Wwox的小鼠(Wwoxfl/fl Lck-Cre)進行實驗。在實驗中將小鼠的脾臟取出並製作成splenocyte suspension且利用抗體刺激T細胞並觀察。透過propidium iodide staining的分析方式去探討細胞的情況。結果顯示刺激T刺激60小時後,Wwox-/- 有較多細胞的死亡因此我的研究中發現了WWOX對於T細胞調控的活化反應,似乎是扮演一種調控性的角色而能夠維持免疫反應的平衡。另外也發現WWOX在活化的T細胞中對於ROS的調控也扮演了重要的清除者。而針對WWOX對於Lck的調控,以及如何清除ROS則需要進一步的探討。
T cell-mediated responses are a central component in immune system for eliminating pathogens and malignant cells. T cell receptor (TCR) signaling is crucial for T cell activation, proliferation, and differentiation. We recently showed that tumor suppressor WW domain-containing oxidoreductase (WWOX) plays an important role in thymocyte development. However, whether WWOX controls TCR signaling for regulating T cell activation remains unknown. By propidium iodide staining, after 60-hr stimulation, increased cell death were observed in Wwox-/- splenocytes as compared with Wwox+/+ splenocytes. Similar results were examined using a T cell-specific Wwox knock-out mouse model. To further investigate the molecular mechanism by which WWOX regulates TCR signaling, protein phosphorylation after TCR triggering was detected in mouse splenic T cells and human Jurkat T cell line. In conclusion, our data suggest that loss of WWOX expression modulates TCR signal for regulation of TCR signaling in Jurkat T cells and cell apoptosis in murine T cells. How WWOX regulates TCR signaling will be delineated in the future.
中文摘要 III
英文摘要 V
Acknowledgement VII
總目錄 VIII
Figure index XII
Introduction 1
TCR-mediated signaling cascade 1
The role of ROS in T cell-mediated response 2
WW domain-containing oxidoreductase 3
Physiological functions of WWOX 4
The role of WWOX in immune system 4
Research objective 5
Materials and Methods 6
A. Materials 6
A-1 Mice 6
A-2 Cell line 7
A-2 Reagents and kits 7
A-3 Antibodies 9
A-4 shRNA clones (purchased from RNAi core) 11
A-5 Consumables 12
A-6 Instruments 13
B. Methods 14
B-1 Cell culture 14
B-2 Preparation of lentiviral shRNA-mediated knockdown cells 16
B-3 Flow cytometric analysis 17
B-4-1 Jurkat T cell seeding and activation 27
B-4-2 Cellular protein extraction 28
B-5 Quantification and adjustment of protein content 30
B-6 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 31
Results 34
Wwox loss causes a transient increase in splenocyte proliferation after anti-CD3 stimulation, whereas reduced proliferative activity and increased cell death are evident after stimulation for 60 hr 34
Aberrant cell proliferation in Wwox-knockout T cells is due to cell intrinsic defect(s) 34
Increased early apoptosis is determined in Wwox-/- mouse splenocytes after anti-CD3 stimulation 35
T cell activation is promptly upregulated in anti-CD3-stimulated Wwox-/- splenocytes, but declined dramatically after stimulation for 60 hr 35
Comparable levels of CD3 and CD28 expression in Wwox+/+ and Wwox-/- splenic T cells 35
Aberrant cell proliferation in Wwox-knockout T cells is not due to defective CD28-mediated co-stimulatory signal 36
Protein phosphorylation of Akt, ERK and NF-B is increased transiently in Wwox-deficient mouse splenic T cells after anti-CD3 stimulation for 15 min, whereas downregulated after stimulation for 24 hr 36
Phosphorylation of proteins in ERK, Akt-mTORc and NF-B pathways is increased transiently in WWOX-knockdown Jurkat T cells after anti-CD3 stimulation within 1 hr, but is downregulated after stimulation for 24 hr 37
Lck activation is increased in WWOX-deficient Jurkat T cells compared to the control cells 37
ROS accumulate more rapidly in Wwox-/- splenocytes after anti-CD3 stimulation for 36 hr as compared to Wwox+/+ splenocytes 38
The mitochondrial membrane potential (ΔΨm) is downregulated evidently in Wwox-deficient splenocytes after anti-CD3 stimulation for 36 hr 38
Phosphorylation of proteins in TCR signaling transiently increases in WWOX-knockdown Jurkat T cells after stimulation within 1 hr is ROS-independent 39
The downregulation of TCR signaling in WWOX-knockdown Jurkat T cells after anti-CD3 stimulation for 24 hr is ROS-dependent 39
The cell proliferation defect and increased cell death in Wwox-deficient mouse splenocytes after anti-CD3 stimulation for 60 hr are rescued by ROS elimination 40
Discussion 41
WWOX acts as a gatekeeper to regulate cell cycle progression in proliferating cells 41
The role of WWOX in alleviating excessive ROS generated in the activated immune cells 41
The regulatory role of WWOX in TCR proximal signaling 42
The regulatory role of WWOX in CSK expression 42
WWOX is crucial for the regulation of mitochondrial function in cells 43
The mitogen-activated protein kinase (MAPK) upstream signaling may be differentially regulated by WWOX after TCR stimulation in T cells 44
Treatment of NAC reduces cell proliferation of Wwox+/+ splenocytes after anti-CD3 stimulation for 60 hr 44
Defective thymocyte development in Wwox knockout mice is probably caused by strong TCR signaling 45
The role of WWOX in autoimmune diseases 46
Conclusion 47
References 48
Figures 56
Appendix 79
Appendix 1. Schematic diagram of TCR signaling cascades 79
Appendix 2. WWOX gene and protein 80
Resume 81
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