臺灣博碩士論文加值系統

根據台灣衛福部106年癌症死亡人數比統計，罹患胃癌的女、男性皆居第六名。當胃受幽門螺旋桿菌感染進入慢性發炎階段，常被認為是導致癌症發病機制的重要因素，其中相關的發炎蛋白質環氧化酶-2 (Cyclooxygenase-2, COX-2)已被證實會於感染過程被激活，且大量表現於臨床胃癌患者檢體中，提升了胃癌的轉移與病程的發生。倒地蜈蚣 (Helminthostachys zeylanica)，富含類黃酮化合物，具保肝、抗發炎、抗氧化等作用，在台灣坊間會用於緩解發燒症狀和發炎性疾病及治療由骨關節炎引起之相關疾病。但目前尚未有倒地蜈蚣應用於胃癌上的相關研究，因此本研究將探討倒地蜈蚣是否可應用於胃癌治療中。我們利用AGS、BGC823等胃癌細胞，以MTT assay、Colony assay、Cell count、Migration assay、 Annexin V staining等實驗觀察藥物對癌細胞存活、增生、遷移、凋亡等影響。使用Real-time polymerase chain reaction (PCR)、Western blotting、Enzyme-Linked immunosorbent Assay (ELISA)等試驗分析發炎蛋白的表現。由結果發現，倒地蜈蚣可有效抑制癌細胞存活、增生、遷移並提升細胞凋亡現象，且可透過抑制COX-2及下游PGE2之表現，減少發炎反應。

According to 2017 statistics of the Taiwan's Ministry of Health and Welfare, the number of deaths among men and women caused by gastric cancer ranked sixth in cancer-related death. When the stomach is infected with Helicobacter Pylori, it goes into a chronic inflammatory phase, which is often considered to be an important factor in the pathogenesis of cancer. The related inflammatory protein Cyclooxygenase-2 (COX-2) has been confirmed to be activated during the infection process, and is abundantly expressed in gastric cancer specimens. Therefore, the metastasis and the course of the gastric cancer are promoted in patients. Helminthostachys zeylanica (H. zeylanica), a flavonoid compound, has the effects of liver protection, anti-inflammation, anti-oxidation. It has been used to relieve fever and inflammatory diseases and treat diseases caused by osteoarthritis in Taiwan. However, it has not been used for the treatment of gastric cancer. Therefore, this study will use the extract of H. zeylanica as a therapeutic drug to explore whether it can be used for the treatment of gastric cancer. We observed the effects of drugs on survival, proliferation, migration and apoptosis of cancer cells by MTT assay, colony assay, cell count, migration assay and annexin V staining in gastric cancer cells (AGS and BGC823).The experssion of inflammatory protein was analyzed by real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting. It was found that H. zeylanica can effectively inhibit the survival, proliferation, migration and apoptosis of cancer cells, and can reduce the inflammatory response by inhibiting the expression of COX-2 and downstream PGE2.

目錄
長庚科技大學學位論文授權書
碩士學位論文指導教授推薦書
碩士學位論文口試審定書
致謝
中文摘要 i
Abstract ii
英文縮寫表 iv
目錄 v
圖目錄 x
附圖目錄 xii
第一章　前言 1
　第一節　背景 1
壹、 人類胃生理功能 1
貳、 人類胃炎及胃癌發生之病理機轉 1
參、 胃癌臨床治療 2
肆、 細胞凋亡 2
伍、 細胞自噬 3
陸、 細胞移行/侵襲 3
柒、 發炎相關蛋白:TNF-α、COX-2、PGE2等與胃癌的影響 4
　第二節　倒地蜈蚣之介紹 5
第二章　研究動機與目標 6
　第一節　研究動機 6
　第二節　研究目的 8
第三章　實驗材料與方法 12
　第一節　溶劑與試劑 12
　第二節　細胞株及培養方法 (Cell lines and cell culture) 13
　第三節　細胞存活 (Cell survival) 14
　第四節　細胞內RNA含量分析 (Intracellular RNA content analysis) 14
壹、 細胞內全部RNA萃取 (Total RNA extraction) 14
貳、 反轉錄聚合酶鏈鎖反應 (RT) 15
參、 DNA分析-即時定量聚合酶連鎖反應 (Real-time PCR) 15
　第五節　細胞內蛋白質分析 (Intracellular protein content analysis) 16
壹、 蛋白質萃取 (Protein extraction) 16
貳、 蛋白質分析-西方墨點法 (Western blotting) 17
　第六節　細胞外PGE2分析 17
　第七節　細胞增生分析 (Cell proliferation analysis) 18
壹、 細胞聚落試驗 (Colony assay) 18
貳、 細胞數目計算分析 (Cell count assay) 18
　第八節　細胞遷移分析 (Cell migration analysis) 19
　第九節　細胞侵襲分析 (Cell invasion analysis) 19
　第十節　流式細胞儀 (Flow Cytometry) 20
壹、 細胞凋亡- Annexin V/7-AAD染色分析 (Annexin V/PI staining assay) 21
貳、 細胞凋亡- Caspase 3/7 染色分析 (Caspase 3/7 staining assay) 21
參、 細胞週期染色分析 (Cell cycle assay) 21
　第十一節　統計分析 (Statistical analysis) 21
第四章　實驗結果 22
　第一節　探討H. zeylanica對於人類胃上皮細胞GES-1及胃癌細胞AGS、BGC823細胞株之細胞存活的影響 22
　第二節　探討H. zeylanica-E2是否能抑制AGS、BGC823的細胞增生情形 23
　第三節　探討H. zeylanica-E2是否具抑制胃癌細胞的移行/侵襲現象 23
　第四節　探討H. zeylanica-E2是否能促使胃癌細胞凋亡 24
　第五節　探討H. zeylanica-E2對人類胃癌細胞週期之影響 26
　第六節　探討H. zeylanica-E2是否能抑制AGS及BGC823發炎因子COX-2之表現 26
　第七節　H. zeylanica-E2抑制人類胃癌細胞中發炎因子PGE2含量 28
　第八節　cPLA2於人類胃癌細胞嚴重發炎時，促使COX-2表現 28
　第九節　探討H. zeylanica-E2是否透過抑制COX-2促使胃癌細胞凋亡/自噬 29
　第十節　探討H. zeylanica-E2是否透過抑制COX-2促使胃癌細胞增生 30
　第十一節　探討H. zeylanica-E2是否透過抑制COX-2促使胃癌細胞移行 31
第五章　討論 32
第六章　結論 42
圖表 45
參考文獻 76
附圖 83

圖目錄
圖一、H. zeylanica 對於GES-1及AGS、BGC823細胞株之細胞存活的影響 45
圖二、H. zeylanica-E2抑制人類胃癌細胞增生 47
圖三、H. zeylanica-E2抑制人類胃癌細胞移行情形 49
圖四、H. zeylanica-E2抑制人類胃癌細胞侵襲情形 52
圖五、H. zeylanica- E2促使人類胃癌細胞凋亡之現象-1 54
圖六、H. zeylanica-E2促使人類胃癌細胞凋亡之現象-2 55
圖七、H. zeylanica- E2促使人類胃癌細胞凋亡之現象-3 57
圖八、H. zeylanica-E2對人類胃癌細胞週期之影響，停滯於G0/G1期 59
圖九、H. zeylanica-E2對人類胃癌細胞中COX-2及PGE2之影響 62
圖十、人類胃癌細胞處於嚴重發炎時，H. zeylanica-E2對COX-2表現之影響 63
圖十一、人類胃癌細胞嚴重發炎時引發cPLA2調控COX-2之表現 64
圖十二、H. zeylanica-E2透過抑制COX-2進而影響人類胃癌細胞自噬相關蛋白及促增生蛋白表現 65
圖十三、H. zeylanica-E2透過抑制COX-2進而影響人類胃癌細胞週期相關蛋白及細胞凋亡蛋白表現 67
圖十四、H. zeylanica-E2透過抑制COX-2進而影響人類胃癌細胞自噬相關蛋白及促增生蛋白表現 69
圖十五、H. zeylanica-E2透過抑制COX-2進而影響人類胃癌細胞週期相關蛋白及細胞凋亡蛋白表現 70
圖十六、H. zeylanica-E2抑制人類胃癌細胞增生 72
圖十七、H. zeylanica-E2抑制人類胃癌細胞移行情形 73
圖十八、H. zeylanica-E2抑制人類胃癌細胞移行情形 74
圖十九、H. zeylanica- E2對胃癌細胞影響示意圖 75
 
附圖目錄
附圖1.全球各類癌症患者總數 83
附圖2.全球各類癌症患者死亡總數 83
附圖3.台灣各類癌症患者死亡人數結構比 84
附圖 4.幽門螺旋桿菌透過NF-κB路徑誘導TNF-α表現示意圖 84
附圖5.細胞凋亡內、外途徑機制示意圖 85
附圖6.Cleaved-PARP促使細胞凋亡示意圖 85

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