臺灣博碩士論文加值系統

胃癌是世界上常見的惡性腫瘤之一，有關胃癌癌化之分子機制仍混沌不清。引發胃癌之危險因子包括飲食、遺傳、幽門螺旋桿菌等，由於胃癌早期症狀不明顯，且易與胃潰瘍之病徵混淆，因此大多病患被診斷出胃癌，已是中後期之癌症，且有伴隨轉移到淋巴或其他器官，如肝臟、腹膜擴散等。對於胃癌的治療方式以摘除胃部組織及周邊淋巴腺廓清為主，合併放射治療及化學藥物治療，但其擴散及轉移之情形，仍導致胃癌之預後不佳。因此研究胃癌癌化之分子機制，作為早期診斷、分期病變及用藥方針現今的重要使命。Notch訊息路徑調控許多細胞內作用，包括增生、分化、凋亡、幹細胞之維持等，對腫瘤生成具有促進或抑制的影響。先前研究指出Notch1訊息路徑可藉由提升COX-2之表現促進胃癌癌化之過程。哺乳類有四種Notch受體蛋白，在結構上具有高度保留的現象，截至目前為止，不同的Notch訊息路徑對於胃癌癌化過程調控機制尚未明瞭。本論文探討Notch3訊息路徑在胃癌癌化過程中所扮演的角色。利用Tet-on系統建立可受doxycycline誘發而大量表現Notch3受體細胞內區域 (N3IC)於SC-M1人類胃腺癌細胞株。經由doxycycline誘發N3IC表現後，SC-M1胃腺癌細胞之增生能力受到抑制，且細胞中cyclin D1、cyclin D3、cyclin E、p21、p53等細胞週期調控蛋白表現量有提升之現象，然而以報導基因表現分析及蛋白質降解分析，發現N3IC可以增加p21轉錄活性並維持其蛋白穩定度。在腫瘤形成能力方面，SC-M1胃腺癌細胞誘發N3IC表現後，細胞群落形成能力被降低，以及皮下注射後之腫瘤生長受到抑制。此外，進一步研究發現SC-M1細胞之移行和侵襲能力也被N3IC所抑制。另一方面，利用AGS和NUGC-3胃癌細胞株進行移行和侵襲能力之分析，也同樣觀察到這些癌化之能力皆被N3IC所抑制。在調控下游基因功能方面，N3IC可以活化c-myc啟動子，並且其轉錄起始點P2區域扮演重要角色。總而言之，這些結果顯示Notch3訊息路徑抑制胃癌癌化過程。

Gastric carcinoma is one of the most common cancers and lethal malignancies worldwide. So far, the molecular mechanisms are still unclear. Salty foods, family history, and Helicobacter pylori infection are seem to higher rick of gastric cancer. The symptoms of early gastric cancer are similar to gastric ulcer or other stomach problems. Most patients were diagnosed with nearby lymph nodes metastasis or in late stage. Surgery followed by chemotherapy or radiotherapy is the most common treatment of gastric cancer. But the spread of lymph nodes and other organs frequently causes poor prognosis of gastric cancer. Therefore study of the molecular mechanism of gastric cancer is very important in modern medicine in the cause of diagnosis, pathogenesis and therapeutic strategies. Notch receptors may regulate cell functions such as proliferation, differentiation, apoptosis and stem cell maintenance. Notch receptors play important roles to modulate tumorigenesis as oncogenes or tumor suppressors. The activation of Notch1 signal pathway promotes gastric cancer progression through COX-2. The four mammalian Notch receptors (Notch1–4) are highly conserved type I transmembrane glycoproteins. The roles of distinct signalings activated by different Notch receptors are still poorly understood in gastric cancer progression. Herein, we sought to address whether Notch3 signaling is involved in control of gastric cancer progression. The Tet-On system expressing Notch3 receptor intracellular domain (N3IC), the activated form of Notch3 receptor, was established in human stomach adenocarcinoma SC-M1 cells. After treatment with doxycycline to induce N3IC expression, proliferation was inhibited in SC-M1 cells. Expressions of cell cycle-regulatory proteins were elevated by N3IC in SC-M1 cells such as cyclin D1, cyclin D3, cyclin E, p21 and p53. Furthermore, N3IC promoted p21 transcription activity and protein stability in SC-M1 cells. N3IC attenuated the xenografted tumor growth of SC-M1 cells. Colony formation, migration, and invasion abilities of SC-M1 cells were suppressed by N3IC. Migration and invasion abilities of other gastric cancer cells were also suppressed by N3IC. Moreover, N3IC enhanced transcriptional activity of Notch downstream target gene c-Myc possibly through P2 transcription start site. Taken together, these results suggest that Notch3 signal pathway suppresses gastric cancer progression.

摘要 i
Abstract ii
縮寫表 iv
目錄 vi
前言 1
胃癌 1
Notch訊息路徑 3
材料 10
細胞株 10
實驗室建立之細胞株 10
質體 11
抗體 12
藥品及試劑 14
器材與儀器 16
引子 17
方法 18
一、細胞培養 18
細胞解凍活化 18
細胞繼代培養 18
細胞計數 19
細胞冷凍保存 20
二、西方點墨法 20
萃取細胞總蛋白質及定量 20
硫酸十二酯鈉聚丙烯醯胺凝膠 21
蛋白質電泳 21
蛋白質轉漬與免疫染色 22
化學發光偵測膜上蛋白 22
三、即時定量聚合酶鏈鎖反應 22
核醣核酸萃取 22
核醣核酸定量 23
核醣核酸反轉錄為去氧核醣核酸 23
即時定量聚合酶鏈鎖反應 24
四、短暫轉染 24
Electroporation短暫轉染法 24
SuperFect短暫轉染法 25
五、報導基因分析 25
六、細胞存活測試 26
七、細胞聚落形成能力試驗 26
八、老鼠移植腫瘤細胞模式 27
九、細胞移行能力試驗及侵襲能力試驗 27
細胞移行能力試驗 27
細胞侵襲能力試驗 28
十、蛋白質降解試驗 29
十一、統計分析 29
結果 30
一、 偵測不同人類胃腺癌細胞株內生性活化態Notch3受體蛋白表現 30
二、 誘導表現活化態Notch3受體之SC-M1胃腺癌細胞確實可以活化Notch訊息路徑 30
三、 表現活化態Notch3受體降低SC-M1胃腺癌細胞生長 31
四、 表現活化態Notch3受體可以調控p21轉錄活性以及蛋白穩度 31
五、 表現活化態Notch3受體抑制SC-M1胃腺癌細胞腫瘤形成能力 32
六、 表現活化態Notch3受體抑制SC-M1胃腺癌細胞轉移能力 32
七、 活化態Notch3受體對上皮間質細胞轉型重要標誌蛋白不具有顯著調控作用 33
八、 活化態Notch3受體可活化c-Myc轉錄活性 33
九、 於AGS及NUGC-3胃癌細胞株中表現活化態Notch3受體皆會抑制轉移之能力。 34
討論 35
參考資料 40
圖表與圖表說明 53
圖一、人類胃腺癌細胞株內生性活化態Notch3受體蛋白表現量 53
圖二、以doxycycline誘導SC-M1/N3IC-myc-His細胞株表現活化態Notch3受體 54
圖三、SC-M1胃腺癌細胞誘導表現活化態Notch3受體後可以活化細胞內Notch訊息路徑 55
圖四、表現活化態Notch3受體降低SC-M1胃腺癌細胞生長 56
圖五、表現活化態Notch3受體影響SC-M1胃腺癌細胞不同的細胞週期調控蛋 57
圖六、表現活化態Notch3受體可以調控p21轉錄活性以及蛋白穩定度 59
圖七、表現活化態Notch3受體抑制細胞群落形成能力 60
圖八、表現活化態Notch3受體抑制SC-M1胃腺癌細胞於裸鼠皮下腫瘤形成能 61
圖九、表現活化態Notch3受體抑制SC-M1胃腺癌細胞轉移能力 62
圖十、表現活化態Notch3受體促進SC-M1胃腺癌細胞傷口癒合能力 64
圖十一、表現活化態Notch3受體可抑制不同胃癌細胞轉移能力 65
圖十二、表現活化態Notch3受體使SC-M1細胞型態較為狹長 66
圖十三、表現活化態Notch3受體不影響SC-M1細胞的上皮間質細胞轉型重要標誌蛋白之表現 67
圖十四、表現活化態Notch3受體可以顯著活化c-Myc啟動子區域轉錄活性 68
圖十五、表現活化態Notch3受體皆可活化不同c-myc啟動子片段轉錄活性 69

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