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研究生:陳螢翎
研究生(外文):Ying-Ling Chen
論文名稱:紫珠水萃物改善尿毒素誘導人肺腺癌細胞的上皮間質轉化之影響
論文名稱(外文):Effects of Callicarpa water extracts Ameliorate Uremic Toxins-induced epithelial-mesenchymal-transition in human lung adenocarcinoma cells
指導教授:徐慧雯徐慧雯引用關係
指導教授(外文):Huey-Wen Shyu
口試委員:王怡棻方麗雯
口試委員(外文):Yi-Fen WangLi-Wen Fang
口試日期:2020-07-14
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:醫學檢驗生物技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:73
中文關鍵詞:紫珠上皮間質轉化肺腺癌A549細胞
外文關鍵詞:Callicarpa sp.epithelial-mesenchymal transitionlung adenocarcinoma A549 cells
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肺癌是世界上最廣泛的癌症之一,因早期診斷率低及易轉移而造成了高死亡率。當癌症開始轉移時,癌細胞會產生上皮-間質轉化 (epithelial-mesenchymal transition, EMT) 。當EMT進行中,細胞也會和週遭的細胞外基質 (extracellular matrix, ECM) 發生交互作用,並使細胞能夠穿越胞外組織移動到其他部位。硫酸吲哚酚 (Indoxyl sulfate, IS) 是眾多尿毒素 (Uremic toxin) 中的一種。高濃度的IS不僅促進慢性腎臟病變的惡化,而且會透過血液循環影響其他器官。研究證實IS可誘導血管內皮細胞發炎反應,促使腎小管細胞EMT,進而導致腎細胞癌。紫珠是一種天然中草藥,其藥理活性包括消炎、抗菌、止血,臨床上常運用在治療風濕、發炎及腸胃道出血等。本研究旨在探究IS是否能參與肺癌細胞的EMT作用,進而造成肺癌的轉移,並進一步探討IS對氧化壓力及發炎反應,並且找出調控的機轉。
本研究結果證實IS能夠促進肺腺癌A549細胞的增殖,可觀察到上皮指標E-鈣黏蛋白 (E-cadherin) 的表現減少,提升間質指標波形蛋白 (Vimentin) 及α-平滑肌肌動蛋白 (α-smooth muscle actin) 的表現量,並證實IS使發炎因子Macrophage migration inhibitory factor (MIF)、細胞激素IL-6等的表現增加,綜合本研究之結果,顯示利用紫珠水萃物處理發現IS可透過NF-κB及Nrf2訊息傳遞路徑調控細胞的EMT與發炎現象。

Lung cancer is the most common cancer worldwide. It is difficult for early diagnosis and causes high mortality due to metastasis. The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell-cell adhesion, and enhance migratory and invasive properties to become mesenchymal cells. Indoxyl sulfate (IS) is one of the circulating uremic toxin. High concentrations of IS will not only promote the deterioration of chronic kidney disease, but also cause damage to endothelium, liver and smooth muscle from the circulation. Many studies have shown that EMT programs can stimulate the production of proinflammatory factors by IS, resulting in renal cell carcinoma. Callicarpa sp. is a Chinese Traditional medicine. Its pharmacological activities include anti-inflammatory, antibacterial, hemostasis and has been applied in the treatment of rheumatism, inflammation and gastrointestinal bleeding.
The aim of this study is to investigate whether IS can participate in the EMT of human lung adenocarcinoma A549 cells, and then promotes the metastasis of lung cancer, and further explores the role of IS for oxidative stress and inflammation, and the underlying mechanism remain unclear.
In this study, we observed that IS treatment induced the cell proliferation of A549 cells in a dose dependent manner. IS-treated cells showed significantly reduced protein expression of E-cadherin and increased protein expression of α-SMA and vimentin in A549 cells. IS-treated cells showed significantly increased protein expression of MIF and IL-6 in A549 cells. In summary, These data suggest that Callicarpa extracts inhibit EMT program, inflammatory cytokine secretion and downregulates NF-κB pathway.

目錄
誌謝…………………………………………………………………...………ii
中文摘要……………………………………………………….…………….iii
英文摘要…………………………………………...……………..…………..v
目錄……………………………………………………...……..…...……….vii
圖目錄……………………………………………………………..…...…….xi
第一章 緒論………………………………………………………………….1
  第一節 肺癌 (Lung cancer)………………………………………….1
    一、肺癌流行病學………………………………………………….1
    二、肺癌的成因………………………………………………….…1
    三、肺癌之分類………………………………………………….…2
  第二節 硫酸吲哚酚 (Indoxyl Sulfate, IS)…………………...………3
  第三節 紫珠水萃物 (Callicarpa sp, Extract)…………..……………5
  第四節 上皮間質轉換(Epithelial-mesenchymal transition, EMT)...7
  第五節 氧化壓力 (Oxidative stress) …………………………….…..9
第六節 Stanniocalcin-2 (STC-2)…………...………....……….……10
研究目的與動機…………………………………………………….…..…..12
實驗設計…………………………………………………………….…..…..13
第二章 材料與方法…………………………...……………………………14
  第一節 實驗材料…………………………………….………………14
    一、實驗用細胞株………………………….……………………14
    二、紫珠熱水溶解法……………………………………………...14
    三、實驗藥品、試劑………………………………….……………14
    四、實驗儀器…………………………………….…………….…15
五、實驗抗體…………………………………….………………..15
  第二節 實驗方法…………………………………………………….16
    一、細胞培養 (Cell Culture) ………………………………….…16
    二、細胞存活率試驗 (Cell Viability Assay) ………………..….17
    三、活性氧物質檢測 (ROS Assay) ……………………….….…18
四、反轉錄聚合酶鏈反應 (Reverse transcriptional polymerase chain reaction , RT-PCR)……………...…………………..….…18
    五、西方墨點法 (Western Blotting Analysis)…………….……20
    六、明膠酶譜法 (Gelatin Zymography Assay)…………………23
    七、傷口癒合試驗 (Wound Healing Assay)……………….……24
    八、Phalloidin & DAPI stain……………………………….……25
    九、質體轉型 (Transformation)………….………………….…26
    十、質體DNA製備 (Plasmid DNA extraction)…………….…26
    十一、短暫性轉染 (Transient transfection)……………..….…27
    十二、冷光酶試驗系統 (Luciferase Assay System)…………….27
    十三、統計分析 (Statistical evaluation)………………….…….28
第三章 研究結果………………………………………………………..….29
  第一節 硫酸吲哚酚對肺腺癌A549細胞生長之影響……………...29
  第二節 硫酸吲哚酚對肺腺癌A549細胞中MIF之影響…………..29
第三節 硫酸吲哚酚對肺腺癌A549細胞中EMT標誌指標及STC-2之影響…………………………………...………………….………...30
  第四節 硫酸吲哚酚與紫珠水萃物對肺腺癌A549細胞生長之影響………………………………………………………………….……30
  第五節 硫酸吲哚酚與紫珠水萃物對肺腺癌A549細胞遷移能力與MMP-2分泌量之影響……………………………………………...…31
  第六節 硫酸吲哚酚與紫珠水萃物所誘導肺腺癌A549細胞EMT標誌表現之影響………………………………………………………….32
  第七節 硫酸吲哚酚與紫珠水萃物對肺腺癌A549細胞中STC-2之影響………………………………………………………..…………...33
  第八節 硫酸吲哚酚與紫珠水萃物所誘導細胞骨架改變之影響.…33
  第九節 硫酸吲哚酚與紫珠水萃物對肺腺癌A549細胞中ROS之影響……………………………………………….………………………34
  第十節 硫酸吲哚酚與紫珠水萃物對肺腺癌A549細胞氧化壓力之影響………………………………………...………..…………………35
  第十一節 硫酸吲哚酚與紫珠水萃物對發炎細胞激素基因表現之調控………………………………………………………………..……...36
第十二節 硫酸吲哚酚與紫珠水萃物對肺腺癌A549細胞中NF-κB訊息途徑之影響………………………………………..………………37
第十三節 硫酸吲哚酚與NAC對肺腺癌A549細胞中ROS及NF-κB訊息途徑之影響………………………………………..…………..38
第十四節 硫酸吲哚酚與NAC對肺腺癌A549細胞中ROS及NF-κB訊息途徑之影響………………………………………..…………..39
第十五節 硫酸吲哚酚與AhR拮抗劑CH223191對肺腺癌A549細胞中ROS及NF-κB訊息途徑之影響………………………………..39
第四章 結論與討論……………………………………………….………40
參考文獻………………………………………………………..…..……….44
附圖………………………………………………………………………….50
附錄一…………………………………………………..…………..……….72
附錄二……………………………………………………...………….…….73

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