根據統計胃癌在全球癌症發生率位居第四名並在癌症相關死亡人數排名達第二名，在胃癌中主要以腺癌(Adenocarcinoma)佔發生總數的90-95%，一般可依照Lauren’s分類以組織學分方式將胃癌分成兩大類，分別是腸型胃腺癌(Intestinal-type Adenocarcinoma)及瀰漫型腺癌(Diffuse Adenocarcinoma)。目前胃癌治療困難的原因在於發現的時期太晚，大多發現時已經是末期。而胃癌高度的抗細胞凋亡與染色體不穩定也造成了臨床上藥物治療的困難，因此了解胃癌發病機制中涉及的機制非常重要的，最近的分子病理學研究已經闡明胃癌是累積的基因組損傷的結果，影響癌症發展所必需的細胞功能（例如，生長信號自足，逃避抗生長信號，凋亡抵抗，持續複製潛能，血管生成誘導以及侵襲性或轉移潛能），胃癌的起始與發展和KRAS、E-cadherin、P53、APC及PTEN具有相關性。
我們實驗室中建立了HK-Cre LKB1L/L PTENL/L和HK-Cre LKB1L/L PTENL/L P53L/L小鼠模型，利用Cre重組酶特異性消除胃部LKB1，PTEN和P53的功能。並且發現HK-Cre LKB1L/L PTENL/L P53L/L小鼠模式會有較高機率發生遠端轉移及周邊組織的侵襲，接著我們利用次世代定序(Next Generation Sequencing , NGS)的方式去分析兩者間差異，發現在HK-Cre LKB1L/L PTENL/L P53L/L小鼠胃癌細胞中會高度表現Twist1。Twist1是一種構型為基本螺旋-環-螺旋（basic helix-loop-helix, bHLH）的轉錄因子，在胚胎發育中多個階段皆扮演的重要的角色，並且對腫瘤轉移能力、腫瘤起始和原發性腫瘤生長都有顯著的相關性。在這項研究中，我們發現Twist1可以誘導胃癌細胞發生上皮 - 間質轉化（EMT）進而增加細胞活體外的運動能力也表示 Twist1亦可促進活體內胃癌細胞對周邊組織的侵襲性及提升遠端轉移的機率。

Gastric cancer is the second leading cause of cancer death worldwide, although the incidence rates is bound by regional geographic variations. More than 95% of gastric cancers are adenocarcinomas developed from the glandular epithelium of the stomach lining. In clinical, a large number of patients are diagnosed when the tumor reached at unresectable late stage. Therefore, it is very important to understand the molecular and pathogenic mechanisms for the development of gastric cancer. Recent molecular pathology studies have elucidated a detailed profile of genetic alterations associated with gastric cancer initiation and progression, which includes activation KRAS and inactivation or loss of E-cadherin, P53, APC and PTEN. Our lab have successfully generated H/K+ATPase–Cre (HK-Cre) LKB1L/L PTENL/L and HK-Cre LKB1L/L PTENL/L P53L/L gastric carcinoma mouse models, which directs the expression of Cre recombinase specific in parietal cells of the stomach to abrogate LKB1,PTEN and P53 function. We first confirmed that our murine gastric carcinoma models develop metastatic gastric carcinomas, histologically similar to human gastric intestinal type carcinoma. In addition, we observed that the metastatic capacity in the HK-Cre LKB1L/L PTENL/L P53L/L mouse model is significantly higher than HK-Cre LKB1L/L PTENL/L model. We further compared gene expression between these two types mouse stomach tumors cell by using Next Generation Sequencing (NGS) analysis, Notably, we found Twist1 is significantly increased expression in the HK-Cre LKB1L/L PTENL/L P53L/L tumor tissue compared to HK-Cre LKB1L/L PTENL/L and wild type. Twist1 is a basic helix-loop -helix (bHLH) transcription factor which plays pivotal roles in multiple stages of embryonic development, inflammation and tumor progression. Ultimately, we demonstrated that Twist1 promotes epithelial-mesenchymal transition (EMT) to enhance gastric cancer cell motility in vitro which may contribute to the metastatic dissemination of cancer cells and the formation of metastasis in vivo.

國立中山大學研究生論文審定書 i
中文摘要 ii
Abstract iii
縮寫表 v
第一章 緒論 1
1.1 胃癌 1
1.2 胃癌動物模式建立 2
1.3 LKB1基因在癌症中的角色 3
1.4 PTEN基因在癌症中的角色 4
1.5 TP53基因在癌症中的角色 4
1.6 Twist1轉錄因子在癌症中的角色 5
第二章 材料與方法 6
2.1 胃癌動物模式 6
2.2 小鼠尾巴DNA萃取 6
2.3 聚合酶連鎖反應 6
2.4 小鼠腫瘤組織處理 8
2.5 組織化學免疫染色 8
2.6 BrdU增殖分析 9
2.7 即時逆轉錄聚合酶鏈鎖反應 9
2.8 細胞培養 10
2.9 細胞轉染 11
2.10 細胞增生試驗 11
2.11 流式細胞儀 11
2.12 西方墨點法 12
2.13 傷口癒合試驗 13
2.14 腫瘤細胞侵襲試驗 13
2.15 3D懸吊式液珠試驗 13
2.16 阿利辛蘭AB染色(Alcian Blue stain) 14
2.17 PAS染色法（Periodic Acid-Schiff stain） 14
2.18 Twist1抑制劑試驗 14
2.19 次世代定序(Next Generation Sequencing , NGS) 14
2.20 統計分析 14
第三章 實驗結果 16
3.1在小鼠胃部特異性去除LKB1、PTEN的活性可導致胃癌發生，如再搭配P53的失活可促使胃癌發生轉移 16
3.2 P53缺失後使胃部腫瘤高度表現腫瘤幹細胞標誌、躲避免疫攻擊及抗凋亡能力 18
3.3 P53缺失會促使上皮細胞中胚轉化(epithelial-mesenchymal transition，EMT)發生 18
3.4 HLPP的腫瘤細胞具有腫瘤發生(tumorigenesis)的能力 18
3.5 在P53缺失的小鼠動物模式中會高度表現Twist1，有助於發生增加胃癌發生轉移 19
3.6 Twist1高表現能促進胃癌細胞發生移行及幹細胞能力 20
3.7 Twist1抑制劑能有效抑制HLPP胃癌細胞移行能力 21
第四章 結果與討論 22
實驗結果圖 24
圖一 胃癌小鼠動物模式建立及PCR基因鑑定分析 24
圖二 不同基因型胃癌小鼠的生理變化及其生存率變化表 26
圖三 H+/K+ ATPase-cre特異性表現於小鼠胃部壁細胞中 27
圖四 H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L (HLP)及H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃部會有明顯腫瘤形成 29
圖五 在H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃癌動物模式中可以看到遠端轉移的發生 31
圖六 H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃部腫瘤會高度表現腫瘤幹細胞標誌、躲避免疫攻擊及抗凋亡能力 32
圖七 H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃部腫瘤有明顯上皮細胞中胚轉化(epithelial-mesenchymal transition，EMT)的發生 33
圖八 從H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L (HLP)及H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠中培養胃部腫瘤細胞 34
圖九 比較H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L (HLP)及H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃癌細胞的腫瘤發生(tumorigenesis)的能力 38
圖十 比較H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L (HLP)及H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃癌細胞的蛋白質表現 39
圖十一 比較H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L (HLP)及H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃癌細胞對於化療藥物的敏感性 40
圖十二 H+/K+ ATPase-cre ; LKB1L/L ; PTENL/L ; P53L/L (HLPP)小鼠胃癌動物模式中會高度表現Twist1 43
圖十三 Twist1在人類胃癌病患中的影響 44
圖十四 去除Twist1表現能抑制胃癌細胞移行及幹細胞能力 46
圖十五 去除HLPP內源性Twsit1會使相關基因受到抑制 47
圖十六 Twist1抑制劑能有效抑制HLPP胃癌細胞移行能力 48
參考文獻 49

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