臺灣博碩士論文加值系統

本研究探討丁酸生產菌對於大腸癌細胞的治療效果，基於此，我們選擇多株大腸直腸癌細胞進行測試，實驗方法則分別使用MTT法、PI染色和Annexin V染色來做分析，初步結果顯示菌株具有抑制結大腸直腸癌細胞的潛力。

This study was aimed to investigate the effect of the butyric acid-producing bacterium on colorectal cancer cells. The experiments were conducted with several colorectal cancer cell lines. The MTT assay, the PI staining, and the annexin V staining were applied for the experimental analysis. The preliminary result showed the potential of the producer strain for treatment of colorectal cancer cells.

目錄
摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 xi
第一章 緒論 1
1.1 前言 1
1.2 文獻回顧 4
1.3 研究動機 13
第二章 實驗方法與材料 14
2.1 菌種之儲存與培養 14
2.1.1 菌種保存 14
2.1.2 菌種培養 15
2.2 質體純化 16
2.3 染色體純化(Chromosome purification) 17
2.4 鏈鎖聚合酶反應(Polymerase Chain Reaction, PCR) 18
2.5 膠體電泳法(Gel electrophoresis)及萃取純化DNA片段 20
2.5.1 膠體電泳法(Gel electrophoresis) 20
2.5.2 萃取純化DNA片段 21
2.6 DNA濃度測量 22
2.7 基因重組技術 23
2.7.1 剪切反應(Digestion) 23
2.7.2 連接反應(Ligation) 24
2.8 大腸桿菌勝任細胞(Competent Cell)的製備 24
2.8.1 以化學法制備勝任細胞 24
2.8.2 以電擊穿透法制備勝任細胞 25
2.9 大腸桿菌的轉形作用(Transformation) 26
2.9.1 以化學法製備勝任細胞的轉形 26
2.9.2 以電擊穿透法製備勝任細胞的轉形 27
2.10 菌種建構與丁酸的生產 28
2.10.1 菌種建構 28
2.10.2 丁酸的生產 31
2.11 大腸癌細胞培養 32
2.11.1 細胞株種類 32
2.11.2 細胞株繼代 32
2.11.3 細胞株保存 33
2.12 純丁酸和丁酸發酵上清液與大腸癌細胞致死測試 34
2.12.1 純丁酸和丁酸發酵上清液與大腸癌細胞反應細胞型態觀察 34
2.12.2 純丁酸和丁酸發酵上清液與大腸癌細胞反應後存活率檢測 34
2.13 PI 染色利用流氏細胞儀觀察丁酸發酵上清液對大腸癌細胞反應後細胞週期影響 35
2.14 Annexin v / propidium iodide染色觀察丁酸發酵上清液對大腸癌細胞凋亡測試 36
2.15 西方墨點法(Western Blotting) 37
2.15.1 樣品製備 37
2.15.1 SDS-PAGE & protein transfer 38
2.15.3 冷光呈色 42
2.16 丁酸上清液對大腸癌腫瘤治療動物模組 42
第三章 結果與討論 43
3.1 系統簡介 43
3.2 丁酸生產 45
3.2. 1 E.coli Nissle生產丁酸路徑測試 45
3.2.2 刪除pta基因，增加丁酸的生產 46
3.2.3 利用含10%FBS DMEM medium進行丁酸醱酵 47
3.3 純丁酸和丁酸醱酵上清液與大腸癌細胞致死測試 48
3.3 Propidium Iodide (PI) 染色利用流氏細胞儀觀察丁酸發酵上清液對大腸癌細胞反應後細胞週期影響 56
3.4 Annexin v / propidium iodide染色觀察丁酸發酵上清液對大腸癌細胞凋亡觀察 59
3.5 西方墨點法(Western Blotting) 62
3.6 丁酸上清液對大腸癌腫瘤治療動物模組 69
3.6.1 時間點丁酸上清液效果體重與腫瘤監測 69
3.6.2 腫瘤與器官切片、H&E染色觀察 75
第四章 結論與未來展望 83
參考文獻 86
附錄 93
實驗器材 93
數據分析 94
PCR引子序列 96
抗體資訊 97

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