胰臟癌是現有人類罹患的惡性腫瘤中最致命與最具有高度轉移侵襲性之癌 症且難以診斷以及預後不佳的一種，因此發展胰臟癌的活體研究模式極為重要。 在此研究中，我們利用基因改造小鼠探討活化 Kras 所誘發的胰臟早期病變並搭 配抑癌基因 APC 和 p53 突變所產生的高度侵襲性胰臟管腺癌 ( PDAC )。在這個 動物模式中，小鼠胰臟在早期就會產生 PanIN( pancreatic intraepithelial neoplasms) 的病變且快速惡化成具高度轉移能力的胰臟腺癌，進一步侵犯其他組織與轉移到 其他器官。在小鼠初代胰臟癌細胞 cDNA 全基因矩陣基因晶片分析中，發現 APC 的突變會增加幹細胞標誌 CD34 蛋白的表現。CD34 為單次跨膜蛋白家族的成員， 其在血管內皮細胞、間質前體細胞和各種間質腫瘤細胞中選擇性表達。然而， CD34 調控胰臟腺演進的詳細分子機轉及影響程度仍然是未知。因此在本論文將 探討 CD34 如何影響胰臟癌惡化的分子機轉。根據本研究的數據顯示，高度表現 CD34 能夠誘導胰臟癌的侵襲和遷移，進而導致腫瘤的轉移。總之，我們的研究 提供了第一線證據，用於描述 PDAC 中 CD34 和 APC / Wnt 通路之間的關聯，並 發現 CD34 在 PDAC 進展中的潛在作用。

Pancreatic cancer is one of the most lethal malignancies because of late diagnosis and limited response to chemo drugs treatment. In this study, we investigated the effects of concomitant p53 and APC mutation on pancreatic neoplasms driven by oncogene Kras in genetically modified mice (GEMM). In this GEMM setting, APC haploinsufficiency coupled with p53 deletion and KrasG12D activation resulted in an earlier appearance of PanIN(pancreatic intraepithelial neoplasms) lesions and progressed rapidly to highly invasive and metastatic pancreatic ductal adenocarcinoma (PDAC). Through the microarray analysis from murine PDAC cells derived from our PDAC models, we observed that the inactivation of APC leads to the upregulated CD34 expression in PDAC. CD34 is a member of a family of single-pass transmembrane proteins, which is selectively expressed in hematopoietic progenitor cells, vascular endothelial cells, interstitial precursor cells and various interstitial tumor cells. However, the functional roles of CD34 in pancreatic cancer remain unclear. Here in, we explored the underlying mechanisms for how CD34 promotes the deterioration of pancreatic malignancy. Our results demonstrated that the increased expression of CD34 induces the invasion and migration of PDAC cells, which may relate to PDAC metastasis in vivo. In summary, our study here provides the first line of evidence to delineate the association between CD34 and APC/Wnt pathway in PDAC, and discover the potential roles of CD34 in PDAC progression.

國立中山大學研究生學位論文審訂書 i
中文摘要 ii
Abstract iii
縮寫對照表 iv
目錄 v
圖次 vii
表次 viii
1.1 胰臟癌 1
1.2 Wnt/β-catenin 信號通路 2
1.3 APC 2
1.4 基因工程小鼠模型 2
1.5 CD34 3
第二章 材料與方法 4
2.1 動物模式 4
2.2 小鼠 DNA萃取 4
2.3 基因型鑑定 5
2.4 組織染色 6
2.5 免疫組織化學染色 6
2.6 細胞株培養 7
2.7 即時定量聚合酶鏈鎖反應 7
2.8 MTT 細胞增生試驗 7
2.9 細胞群落分析 8
2.10 傷口癒合試驗 8
2.11 細胞侵襲試驗 8
2.12 腫瘤球體形成試驗 9
2.13 流式細胞術 9
2.14 西方墨點法 9
2.15 cDNA 微陣列分析 11
2.16 細胞免疫螢光染色 12
2.17 細胞轉染 12
2.18 統計分析 13
第三章 實驗結果 14
3.1 APC 和 p53 缺失與活化突變體 KrasG12D 會導致胰臟癌的發生 14
3.2 PKAP+ 腫瘤細胞會促進腫瘤生成以及細胞遷移的能力 14
3.4 PKAP+ 腫瘤細胞會誘導EMT使腫瘤細胞具有轉移能力 15
3.5 PKAP+ 腫瘤細胞高度表現幹細胞標誌 CD34 16
3.6 過度表現 CD34 會增加腫瘤生成和細胞遷移的能力 17
3.8 PKAP+ 腫瘤細胞中降低 CD34 表現會抑制細胞侵襲和遷移的能力 18
3.9 PKP+ 腫瘤細胞過度表現CD34在體內試驗具有高度轉移能力 18
第四章 結果與討論 19
實驗結果圖 21
參考文獻 41
附錄 44

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