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研究生: 林芷彤
Lin, Chih-Tung
論文名稱: Ⅰ、SCNN1B於大腸直腸癌類癌幹細胞之角色探討 / Ⅱ、建立藥物吸收之活細胞即時影像觀測系統
Ⅰ、The role of SCNN1B in stemloid of colorectal cancer / Ⅱ、Establishment of Cellular Live-Imaging Model for drug Absorption
指導教授: 賴韻如
Lai, Yun-Ju
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2018
畢業學年度: 106
語文別: 中文
論文頁數: 70
中文關鍵詞: 大腸直腸癌癌幹細胞類癌幹細胞標靶治療SCNN1B口服給藥藥物吸收Caco-2薑黃素
英文關鍵詞: Colorectal cancer (CRC), cancer stem cells (CSCs), cancer stemloids, target therapy, SCNN1B, Oral administration, Drug absorption, Caco-2, Curcumin
DOI URL: http://doi.org/10.6345/THE.NTNU.SLS.018.2018.D01
論文種類: 學術論文
相關次數: 點閱:96下載:5
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  • Ⅰ、大腸直腸癌類癌幹細胞因子之探討
    大腸癌 (colorectal cancer, CRC) 又稱為大腸直腸癌或結腸直腸癌,由於其預後效果並不理想,即便經過治療,仍常發生轉移及復發的情形。「癌幹細胞 (cancer stem cells, CSCs)」為腫瘤細胞中極少部分類似幹細胞特性的細胞。然而這些細胞與腫瘤的發生、轉移、抗藥性以及復發擁有密切的關係。目前常用於辨識癌幹細胞的生物標記,如:CD133、CD44等,但這些生物標記仍備受爭議。本研究中我們利用腫瘤病人的新鮮檢體培養得來之貼盤細胞與懸浮細胞球(tumorsphere) 進行微陣列分析 (microarray assay),實驗結果發現SCNN1B基因的表現量在類癌幹細胞中有較一般癌細胞多的趨勢;且在大腸癌細胞株的繼代培養中亦觀察到相同的現象。因此,我們期望透過研究SCNN1B對大腸癌幹細胞 (colorectal cancer stem cells, CRCSCs) 的影響,藉以找出大腸癌治療標靶的新穎生物標記。

    Ⅱ、建立藥物吸收之活細胞即時影像觀測系統
    一直以來,在臨床上給予藥物最常用的方式是口服給藥,因為口服給藥是最為經濟且安全的,並被認為是病患對於藥物吸收最理想的方式,口服給藥的方式藥物會通過腸道吸收以及血管運送進入人體循環,上皮細胞對於藥物的吸收程度以及藥物對於內皮細胞的穿透能力,被認為是對於藥效影響的指標。目前在臨床研究上有許多方法可以用來偵測藥物在體內以及體外的吸收程度,但這些方法大部分是建立在間接的化學分析或者物理波長的吸光度測定上,因此本研究透過建立一個活細胞即時影像系統,觀察藥物吸收以及藥物進入細胞的過程。利用計算藥物螢光的強度,我們可以偵測藥物或生物活性配方的吸收效率,而我們也成功的在加藥半小時內觀察到薑黃素在Caco-2細胞的吸收情形。此外,我們也觀察到經過高速均質後的薑黃素乳化劑吸收的效率比單純脂質包裹的薑黃素來得高。

    Ⅰ、The role of SCNN1B in stemloid of colorectal cancer
    Colorectal cancer (CRC), also known as colon cancer, is the third most common cancer in Taiwan. Its prognosis is poor because of high rates of recurrence and metastasis. Cancer stem cells (CSCs) are few percentages of cells in a tumor which have characteristic of stem cell. These cells are related to tumor initiation, metastasis, drug resistance, and relapse. Some biomarkers of CSCs have been found, such as CD133 and CD44. However, these biomarkers are still controversial. To identify more representative genes in regulation of colorectal cancer stem cells (CRCSCs), we performed microarray analysis and identify SCNN1B that expresses higher in tumor spheres than in adherent cancer cells derived from patients’ fresh specimens . We also observed the same phenomenon in CRC cell lines, HCT116 and DLD-1, and the tumorspheres derived from these cells for three passages. By studying the effects of SCNN1B on CRCSCs, we may identify a biomarker that serves as a new target in cancer therapy.

    Ⅱ、Establishment of Cellular Live-Imaging Model for drug Absorption
    Oral administration is the most common method in taking clinical medicine. It is also the safest, most convenient, and economical way. By oral administration, drug is absorbed by digestive system and enter circulatory system. In the study of drug absorption, it is important to evaluate the relationship of these oral drug formulations and the efficiency of absorption. The level of drug absorption in the epithelial cells and the ability of penetration through the endothelial cell are two important factors that determine the final effects of a drug. Until now, there are many methods to evaluate the drug absorption levels in vitro and in vivo. However, majority of these assays were established on indirect chemical analysis assays or physical wavelength absorbance assay. Therefore, we established a cellular live-imaging model to observe the process of drug absorption and penetration in live cells using real-time microscopy. By measuring the intensity of bio-fluorescence of the drug or a labeled micelle, we can determine the efficiency of the cellular absorption of a specific drug or a formula. Here we successfully observed the curcumin absorption by colon cancer cells Caco-2 in half hour after treatment. Furthermore, the curcumin with high-speed homogenized-emulsion showed the better cellular absorption compared to the lipid-wrapped one.

    目錄 縮寫表 1 Ⅰ、大腸直腸癌類癌幹細胞因子之探討 3 摘要 3 Abstract 4 Ⅰ-1 緒論 5 Ⅰ-1-1 大腸直腸癌 (Colorectal cancer, CRC) 5 Ⅰ-1-2 癌幹細胞假說 (Cancer stem cell theory) 7 Ⅰ-1-3 SCNN1B (Sodium channel epithelial 1 beta subunit) 8 Ⅰ-2 研究材料與研究方法 10 Ⅰ-2-1 細胞培養(Cell culture) 10 Ⅰ-2-1.1 貼盤細胞培養 10 Ⅰ-2-1.2 懸浮細胞培養 10 Ⅰ-2-2 初級細胞培養 (Primary cell culture) 11 Ⅰ-2-3 細胞及組織RNA萃取 (RNA extraction from cell line and tissue) 12 Ⅰ-2-4 RNA定量 13 Ⅰ-2-5 反轉錄聚合酶連鎖反應 (Reverse Transcription Polymerase chain reaction, RT-PCR ) 13 Ⅰ-2-6 細胞蛋白質萃取 14 Ⅰ-2-7 數據資料庫分析 15 Ⅰ-2-8 統計結果分析 15 Ⅰ-3 結果 16 Ⅰ-3-1 SCNN1B其mRNA在檢體培養的類癌幹細胞中表現量高於同為檢體培養之貼盤細胞 16 Ⅰ-3-2 SCNN1B其mRNA在大腸癌細胞株的表現差異 16 Ⅰ-3-3 繼代培養的細胞株,其SCNN1B基因mRNA之差異 17 Ⅰ-3-4 SCNN1B基因在p53突變之細胞株DLD-1的表現量 17 Ⅰ-3-5 SCNN1B基因表現量多寡對於大腸癌病患存活率的影響 18 Ⅰ-4 討論 19 Ⅱ、建立藥物吸收之活細胞即時影像觀測系統 22 摘要 22 Abstract 23 Ⅱ-1 緒論 24 Ⅱ-1-1 口服藥物在體內的吸收 24 Ⅱ-1-2 研究藥物吸收常用之模型 25 Ⅱ-1-2.1 非生物藥物吸收模型 26 Ⅱ-1-2.2 體內藥物吸收模型 26 Ⅱ-1-2.3 體外藥物吸收模型 27 Ⅱ-1-3 藥物吸收之活細胞即時影像觀測系統 28 Ⅱ-2 研究材料與研究方法 29 Ⅱ-2-1 細胞培養 29 Ⅱ-2-2 活細胞即時影像觀測系統模型建立 30 Ⅱ-2-2.1 細胞製備 30 Ⅱ-2-2.2 薑黃素乳化液 30 Ⅱ-2-2-3 共軛焦顯微鏡拍攝 31 Ⅱ-2-3 傳統藥物吸收體外模型 31 Ⅱ-2-3.1 以Transwell培養細胞 31 Ⅱ-2-3.2 跨膜電阻的測量及計算 32 Ⅱ-2-3.3 HPLC樣品的製備 33 Ⅱ-2-4 HPLC 33 Ⅱ-2-4 統計分析 34 Ⅱ-3. 結果 35 Ⅱ-3-1 U373-MG細胞株之前測試驗 35 Ⅱ-3-2 Caco-2細胞株之前測試驗 35 Ⅱ-3-3 Caco-2對於薑黃乳化液之吸收 36 Ⅱ-3-4 傳統Transwell方法實測細胞吸收 38 Ⅱ-3-4.1 傳統藥物研究模型 38 Ⅱ-3-4.2 HPLC之檢測結果 39 Ⅱ-4. 討論 40 III、圖表 44 IV、參考文獻 66

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