臺灣博碩士論文加值系統

肝癌為全球最常見的惡性腫瘤之一，其癌症相關致死率則排名全球第三，目前治療肝癌的方式侷限於肝臟切除與手術移植。肝癌的特性為具高度的血管分佈，能夠幫助腫瘤細胞生長。在肝癌細胞中細胞激素IL-6能夠活化STAT3蛋白。另外，中心體蛋白CPAP除了在中心體扮演重要角色外，亦為NF-B與STAT5的轉錄輔助活化因子 (transcriptional co-activator)。實驗室先前的研究發現，CPAP在B型肝炎病毒感染所致的肝癌病人中有過量表現的情形。本論文主要探討在肝癌中CPAP參與在IL-6/STAT3訊息傳遞途徑中的功能性角色。我們的研究發現，在IL-6刺激下，過量表現GFP-CPAP可增加STAT3轉錄活性及其下游標的基因的表現。在免疫共沉澱 (co- immunoprecipitation)的實驗中也觀察到CPAP可以和STAT3形成複合體。此外，我們也發現，在肝癌檢體中CPAP和p-STAT3的表現呈正相關，此外，根據研究報導指出IL-8會藉由增加血管內皮細胞存活、增生、基質金屬蛋白酶的分泌調控細胞內血管新生。有趣的是，在IL-6刺激下，過量表現GFP-CPAP可增加IL-8的啟動子活性，且在酵素免疫分析法 (ELISA)實驗中也發現，GFP-CPAP能增加IL-8與VEGF的蛋白分泌。利用原位異種移植與脾臟注射動物模式，我們發現CPAP會促進肝癌細胞之生長和轉移。此外，由肝癌檢體也發現CPAP與病人血清中IL-8蛋白的表現呈正相關。這些結果顯示，CPAP可能透過IL-6/STAT3途徑促進IL-8與VEGF的表現而導致肝癌的惡化。

Overexpression of GFP-CPAP significantly enhanced STAT3-driven transcriptional activation in response to IL-6 treatment. Overexpressed GFP-CPAP transcriptionally increased the targeted gene expression of STAT3 upon IL-6 stimulation. Co-immunoprecipitation showed that CPAP can form a complex with STAT3 in response to IL-6 treatment. Immunoblot analysis further indicates that overexpression of CPAP is positively correlated with activated STAT3 (p-STAT3) in HCC tissues. On the other hand, it was reported that interleukin-8 (IL-8) can regulate angiogenesis by enhancing endothelial cell survival, proliferation and matrix metalloproteinases production. Interestingly, overexpression of GFP-CPAP enhances the promoter activity of IL-8 under IL-6 treatment. The enhanced expression of IL-8 and VEGF by overexpressed GFP-CPAP were further confirmed by ELISA. By orthotopic and splenic mouse model, it showed that overexpressed CPAP can promote tumor growth and metastasis. Moreover, the expression of CPAP is positively correlated with plasma IL-8 in HCC patients. Taken together, our results suggest that CPAP may contribute to HCC malignancy through the IL-6/STAT3 pathway-mediated IL-8 and VEGF expressions.

目錄
中文摘要....I
英文摘要....II
誌謝....IV
目錄....VI
圖目錄....XI
附錄目錄....XIII
縮寫檢索表....XIV
第一章 緒論....1
1-1. 肝癌....1
1-2. IL-6 (Interleukin-6)和肝癌的關聯性....2
1-3. STAT蛋白家族 (Signal Transducers and Activators of Transcription Protein Family, STAT)....3
1-3.1 STAT蛋白....3
1-3.2 STAT3 (Signal Transducers and Activators of Transcription Protein 3)的簡介與活化途徑....3
1-3.3 STAT3在腫瘤形成 (Tumorigenesis)所扮演的角色....5
1-4. IL-6/STAT3訊息傳遞途徑與肝癌的關係....6
1-5. CPAP (Centrosomal P4.1-Associated Protein)....7
1-6. 研究動機....8
1-7. 研究目的....9
第二章 實驗材料與方法....10
2-1. 細胞株培養....10
2-1.1 細胞株種類、培養液及培養方式....10
2-1.2 繼代培養 (Subculture)....10
2-1.3 條件培養液備製 (Conditioned Medium Preparation)....11
2-2. 短暫性轉殖感染 (Transient Transfection)....11
2-2.1過量表現 (Overexpression)....11
2-2.2基因默化 (Gene silencing)....12
2-3. 全細胞液抽取 (Total cell Lysate Extraction)....12
2-3.1 RIPA buffer的配製....12
2-3.2 抽取方式....13
2-4. 蛋白質濃度測定....13
2-5. 西方墨點法 (Western Blotting)....14
2-5.1 硫酸十二酯鈉聚丙烯醯胺凝膠 (SDS-PAGE)配製....14
2-5.2電泳 (Electrophoresis)....14
2-5.3濕式轉漬法 (Wet Transfer)....15
2-5.4 封阻步驟 (Blocking)....16
2-5.5 抗體 (Antibody)....16
2-5.6 免疫染色法 (Immunoblotting)....17
2-6. 報導基因分析 (Reporter Assay)....17
2-6.1 報導基因載體 (Reporter plasmid)....17
2-6.2 報導基因分析實驗....18
2-7. 全量RNA抽取 (Total RNA Extraction)....18
2-8. 反轉錄反應 (Reverse Transcription)....19
2-9. 即時定量聚合酶連鎖反應 (Quantitative Real-time Polymerase Chain Reaction, Q-PCR)....20
2-9.1 引子設計....20
2-9.2 實驗方法....21
2-10. 酵素免疫分析法 (Enzyme-Linked Immunosorbent Assay, ELISA)....21
2-11. 免疫共沉澱法 (Co- Immunoprecipitation assasy, Co-IP) ....22
2-12. 原位異種移植動物模式 (Orthotopic Xenograft Model) ....23
2-13. 脾臟注射動物模式 (Spenic Injection Model)....24
2-14. 質體轉殖 (Transformation)....25
2-15. 抽取大量質體DNA (Midi Prep)....25
第三章 結果....27
3-1. CPAP參與在IL-6所誘導的STAT3活化訊息傳遞途徑....27
3-1.1 在肝癌細胞大量表現CPAP會增加IL-6所誘導的STAT3轉錄活性 ....27
3-1.2 大量表現CPAP會增加肝癌細胞在處理IL-6後STAT3下游基因的表現....28
3-1.3 CPAP是透過IL-6/Jak2訊息途徑活化STAT3 ....29
3-1.4 CPAP在IL-6刺激下可以與STAT3形成複合體....29
3-2. 利用原位異種移植與脾臟注射動物模式發現CPAP能促進肝癌細胞的生長與轉移....30
3-2.1 CPAP在原位異種移植動物模式中促進肝癌細胞生長及轉移 ....30
3-2.2 CPAP在脾臟注射動物模式中促進肝臟轉移....31
3-3. 分析臨床肝癌病人檢體發現CPAP的表現量和p-STAT3及病人血清中IL-8的表現量呈正相關....33
3-3.1 在臨床肝癌病人組織中，CPAP與p-STAT3的表現呈正相關....33
3-3.2 在臨床因B型肝炎病毒感染所致的肝癌病人檢體中，CPAP在肝臟腫瘤組織的表現與病人血清中IL-8表現呈正相關....34
3-4. 總結....34
第四章 討論....36
4-1. 探討在IL-6刺激下NF-B和STAT3的角色....36
4-2. 探討CPAP在IL-6刺激下透過何種機制增加STAT3的轉錄活性及活化 ....37
4-3. 探討CPAP在IL-6刺激下的功能性角色....38
4-3.1 探討CPAP是否促進由IL-6所引起之血管新生....38
4-3.2 探討CPAP是否促進由IL-6所引起之細胞增生....39
4-4. 總結與未來方向....39
參考文獻....41
圖目錄
Fig 1. Overexpression of CPAP enhances IL-6-induced STAT3 transcriptional activity....46
Fig 2. Overexpression of CPAP increases the promoter activity of IL-8, ICAM-1 and IL-6 upon IL-6 treatment ....47
Fig 3. Overexpression of CPAP augments the expression of IL-8, ICAM-1, VEGF and HIF-1 mRNA....48
Fig 4. Overexpression of CPAP enhances the expression of IL-8, VEGF and IL-6....49
Fig 5. CPAP increases the transcriptional activity of STAT3 through IL-6/Jak2 pathway....51
Fig 6. CPAP can form complex with STAT3....52
Fig 7. CPAP promotes the tumor growth and lung metastasis of HCC in orthotopic xenograft mouse model....53
Fig 8. CPAP increases liver metastasis in splenic injection mouse model....55
Fig 9. The expression level of human or mouse CPAP and actin in Hep3B cells and mouse liver tissue....57
Fig 10. CPAP and p-STAT3 have positive correlation in clinical HCC tissues....58
Fig 11. Plasma IL-8 is positively correlated with CPAP but not HBx in HBV-HCC patients....60
Fig 12. Proposed model for overexpressed CPAP in IL-6/STAT3 pathway in HCC....61
Fig 13. Knockdown expression of CPAP increases the phosphorylation of STAT3 with IL-6 treatment....62
Fig 14. Crosstalk between STAT3 and NF-B in response to IL-6 or TNF- treatment....63
Fig 15. NF-B inhibits IL-6-mediated STAT3 transcriptional activation in HCC cells but not in colorectal cancer cells....64
Fig 16. Knockdown of p65 increases the effect of CPAP-enhanced STAT3 activation with IL-6 treatment....66
Fig 17. Overexpression of CPAP doesn’t increase cyclin D1 mRNA expression and doesn’t promote IL-6-induced cell proliferation....67
附錄目錄
[附錄一] Amounts for DNA transfection in different culture vessel....68
[附錄二] Amounts for RNAi transfection in different culture vessel....69
[附錄三] pGL4.47[luc2P/SIE/Hygro] vector map....70

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