胃癌是世界最常見的癌症類型之一，它涉及廣泛的局部腫瘤侵襲、轉移和不良的預測後果。為了開發有效的治療策略我們必需了解胃癌進程的調節機制。過氧化物酶體增殖物激活受體-δ（PPARD）在許多主要的人類癌症中是增加的，但是在癌細胞轉移的過程中所扮演的角色仍然所知甚少。本篇論文中，我們發現在胃癌誘導的動物模型中，將PPARD表現量降低或基因缺失能夠顯著地抑制胃癌細胞的轉移。首先，我們發現PPARD的表現量與胃癌的預後不良有關聯性。缺氧的情況下顯著誘導胃癌細胞中PPARD的表現。機制方面，細胞與動物實驗都可以看到PPARD透過調節Snail的表現量促進遷移和轉移。 Snail通過抑制緊密連接蛋白的表現來引發上皮間質轉化（EMT）。PPARD在缺氧條件下促進胃癌細胞遷移，透過基因靜默（shRNA-PPARD）的方式可以逆向證明這個結果。同時我們也發現，在缺氧條件下，缺氧誘導因子1α（HIF1A）直接調節PPARD的量，這代表了正常組織和癌症中缺氧反應的關鍵角色。在缺氧條件下PPARD的上調需要HIF1A。在動物研究中，使用PPARD促進劑可誘導細胞遷移和轉移，抑制劑則降低之。shRNA-PPARD對PPARD的基因沉默完全抑制了上述腹膜轉移的效應。綜合上述所說，我們的結果發現HIF1A是一個新的PPARD表現調控者，並表明PPARD是缺氧誘導胃癌進程的關鍵介質。靶向PPARD表達或活性可以成為一個阻斷侵襲性胃癌進程的有效手段。

Gastric cancer (GC) is one of the most common types of cancer worldwide, and it involved extensive local tumor invasion, metastasis and poor prognosis. Understanding the mechanisms regulating the progression of GC is necessary for the development of effective therapeutic strategies. Peroxisome proliferator–activated receptor–δ (PPARD) is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in GC models in vivo. Firstly, we found that PPARD expression levels dependently associated with poor prognosis in gastric cancer. Hypoxia markedly induced expression of PPARD in gastric cancer cell lines. Mechanistically, PPARD promoted migration and metastasis regulates Snail of expression in vivo and in vitro. Snail triggers the epithelial‐mesenchymal transition (EMT) by repressing expression of tight junction proteins. PPARD promotes cell migration of gastric cancer under hypoxia condition, which could be abolished markedly by gene silencing (shRNA-PPARD) Moreover. we found that under hypoxic conditions, hypoxia-inducible factors 1 α (HIF1A) directly regulated the abundance of PPARD, which are key players of the hypoxia response in normal tissues and cancers. The upregulation of PPARD in hypoxic conditions required HIF1A. In animal study, exposure to PPARD agonists promotes cells migration and metastasis, and vice versa in antagonists. Gene silencing of PPARD by shRNA-PPARD completely inhibited above-mention of peritoneal dissemination effects. Taken together, our results identify HIF1A as novel regulators of PPARD expression and suggest that PPARD is a key mediator of hypoxia-induced gastric cancer progression. Targeting PPARD expression or activity may provide an effective means to block the progression of aggressive gastric cancers.

目次
中文摘要 i
英文摘要 ii
目次 iii
圖目次 v
第一章、前言 1
一、胃癌 1
(一) 流行病學 1
(二) 癌症分期 2
(三) 治療方式 3
二、缺氧 (Hypoxia) 4
三、HIF1A (Hypoxia-inducible factor 1-α) 4
四、PPARD (peroxisome proliferator-activated receptor δ) 4
五、上皮-間質轉化 (Epithelial-mesenchymal transition；EMT) 6
六、研究方向與動機 6
第二章、材料與方法 7
一、實驗儀器 7
二、實驗材料 7
(一) 常用溶液: 附表一 7
(二) 實驗試劑: 附表二 7
(三) 實驗藥品: 附表三 7
(四) 實驗抗體: 附表四 7
(五) PCR Primer: 附表五 7
(六) sh-RNA: 附表六 7
三、實驗方法 8
(一) 細胞培養 (Cell culture) 8
(二) 蛋白質萃取 (Protein extraction) 8
(三) 西方墨點法 (Western Blot；WB) 9
(四) 核酸萃取(RNA extraction) 9
(五) 反轉錄聚合酶連鎖效應(Reverse Transcription-PCR) 10
(六) 聚合酶連鎖效應(Polymerase Chain Reaction) 10
(七) 免疫螢光染色 (Immunofluorescence stain) 11
(八) 蘇木素-依紅染色(Hematoxylin and Eosin Stain) 11
(九) 免疫組織染色 (Immunohistochemistry stain；IHC) 12
(十) 瓊膠生長實驗 (Colony Formation Assay；Soft Assay) 12
(十一)細胞核質蛋白分離萃取(Nuclear/ Cytosol protein extraction) 13
(十二) 免疫沉澱法 (Immunoprecipitation) 13
(十三) 核酸干擾技術 (Small Hairpin RNA) 14
(十四) 傷口癒合試驗 (Wound Healing Assay) 14
(十五) 動物實驗 (Animal experiment) 15
(十六) 動物組織蛋白萃取 15
(十七) 電泳移動率試驗 (EMSA) 15
(十八) 染色質免疫沉澱法 (CHIP Assay) 16
(十九) 流氏細胞儀分析(Flow cytometry) 17
(二十) 細胞增生試驗(MTT Assay) 17
(二十一) 免疫細胞化學檢測缺氧 18
第三章、實驗結果 19
一、在胃癌病人中HIF1A與PPARD過量表達 19
二、在胃癌中缺氧能導致PPARD過量表達 19
三、PPARD表達不影響細胞增殖 20
四、缺氧誘導PPARD增加細胞遷移並增加上皮-間質轉化(EMT)指標蛋白 20
五、HIF1A增加PPARD轉錄表現 21
六、給予PPARD拮抗劑治療與靜默PPARD能抑制裸鼠胃腫瘤生長與腹膜轉移 21
七、結論 22
第四章、討論 23
第五章、未來展望 25
第六章、參考文獻 26
結果圖表........................................................................................................28
附錄表............................................................................................................47

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