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研究生:黃衍筠
研究生(外文):Hin-Kwan Wong
論文名稱:凝血酶調節素透過蛋白激酶A抑制腫瘤細胞生長之機轉
論文名稱(外文):Thrombomodulin suppresses tumor cell proliferation by the inhibition of protein kinase A
指導教授:施桂月
指導教授(外文):Guey-Yueh Shi
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:78
中文關鍵詞:凝血酶調節素腫瘤生長調控
外文關鍵詞:thrombomodulintumorproliferation
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腫瘤細胞生長的調控在癌症生成 (tumorigenesis) 的過程中是非常重要的。凝血酶調節素 (thrombomodulin, TM) 為表現於細胞膜上的第一型膜醣蛋白(type I cell surface glycoprotein),是一種廣為人熟悉的抗凝血因子 (anticoagulant)。臨床證據表示腫瘤細胞上 TM 的減少與許多癌症轉移 (metastasis) 的發生相關,而近年更有研究進一步發現 TM 可抑制腫瘤細胞生長。為了探討 TM 如何調控腫瘤細胞生長,我們深入研究與其產生交互作用 (interaction) 的蛋白,以及其後所影響的訊息傳遞路徑 (signaling pathway)。我們首先證實在腫瘤細胞中 TM 的表現比正常細胞少。當 TM 表現量愈少時,細胞內的蛋白激酶 A (cAMP-dependent protein kinase, PKA)、下游胞外調節激酶 (extracellular signal-regulated kinase, ERK) 的活性、以及腫瘤細胞生長的速率均愈高。我們接著發現 TM 會透過其細胞內結構區 (cytoplasmic domain, TMD5) 與 ezrin 及 PKA 形成複合體 (complex)。而當專一性截短 TMD5 後,細胞內的 PKA 活性亦會大量上升,生長速率亦會變快。此外,在持續活化細胞內的 PKA 活性後,TM 會被磷酸化並同時觀察到這複合體瓦解。根據以上結果,我們提出一個全新的凝血酶調節素調控腫瘤細胞生長的機制:TMD5、ezrin 與 PKA 形成複合體後,會進而穩定 PKA 四合體 (tetramer) 的結構,使其活性下降而無法活化 ERK,造成下游的訊息傳遞被中斷而抑制細胞生長;當腫瘤細胞惡化後、因為 TM 的表現大量下降,使細胞內 PKA 活性持續上升而磷酸化 TMD5,造成更多複合體瓦解,促成正回饋調控環 (positive feedback loop) 使細胞內 PKA 活性更高、腫瘤細胞生長速率更快,故此 TM 的大量表現可幫助抑制腫瘤細胞的生長。
The regulation of tumor cell proliferation is an important issue in tumorigenesis. Thrombomodulin (TM), a type I cell surface glycoprotein, is well characterized as an anticoagulant which functions through thrombin-dependent protein C activation. Recent evidences indicated that TM reduction in tumor cells is associated with various tumor metastases. Furthermore, it was reported that TM downregulates the growth of tumor cells. To clarify the mechanism of TM in regulating tumor growth, we explored the possible interaction proteins and the signaling pathway involved. TM expression was lowered in tumorigenic cancer cells. Loss of TM was associated with higher extracellular signal-regulated kinase (ERK) activation and increased proliferation rate. Cyclic AMP-dependent protein kinase (PKA), which was previously identified to activate ERK, was also activated. We discovered that TM cytoplasmic domain (TMD5) formed a complex with PKA via ezrin. The truncation of TMD5 greatly increased the cellular PKA activities and cell proliferation rate. Moreover, stimulation of PKA induced TMD5 phosphorylation and triggered the complex dissociation. In conclusion, we demonstrated the novel mechanism of TM in suppressing cell growth: TM/ezrin/PKA form a complex to stabilize the PKA tetramer, inactivate PKA and disrupt the downstream cell growth ERK signaling pathway. Therefore, loss of TM in tumor cells upregulates the cellular PKA activities and further induces the TM phosphorylation and complex dissociation. As a result, the positive feedback loop of PKA activation accelerates the tumor cell growth.
中文摘要 I
ABSTRACT II
ACKNOWLEDGEMENTS III
TABLE OF CONTENTS IV
LIST OF FIGURES VI
LIST OF APPENDICES VII
LIST OF ABBREVIATIONS VIII
INTRODUCTIONS 1
1.1 THE STRUCTURE OF THROMBOMODULIN 1
1.2 THE MULTIFUNCTIONAL ROLE OF THROMBOMODULIN 2
1.3 THE HISTORY OF THROMBOMODULIN IN TUMOR BIOLOGY 4
1.4 INTRODUCTION TO PROTEIN KINASE A 5
1.5 THE ROLE OF PROTEIN KINASE A IN TUMOR CELL GROWTH 6
1.6 INTRODUCTION TO A-KINASE ANCHORING PROTEIN 7
1.7 MOTIVATION AND RATIONALE OF THE STUDY 8
MATERIALS AND METHODS 9
2.1 CELL CULTURE 9
2.1.1 Cell line 9
2.1.2 Subcultivation 10
2.1.3 Cell counting 14
2.1.4 Cryogenic preservation and storage of cell cultures 16
2.1.5 Cell morphology image caption 18
2.2 CELL PROLIFERATION ASSAY 18
2.3 GENERAL PROTEIN ANALYSIS 20
2.3.1 Protein sample preparation 21
2.3.2 Protein quantitation 22
2.3.3 Electrophoresis 23
2.3.4 Western blotting analysis 29
2.4 IMMUNOPRECIPITATION 32
2.5 NON-RADIOACTIVE CAMP DEPENDENT PROTEIN KINASE ASSAY 34
2.6 TUMOR GROWTH IN VIVO 38
RESULTS 41
DISCUSSIONS 45
CONCLUSION 48
RECOMMENDATIONS 49
REFERENCES 50
FIGURES 60
APPENDICES 72
AUTHOR PROFILE 78
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