臺灣博碩士論文加值系統

Our previous study on anti-tumor activity of Taiwanofungus cam-phoratus alcohol extract (TCAE) suggested that the treatment of TCAE on HT-29 colon cancer cell line not only promoted apoptosis but also conferred a synergistic effect by pretreating a small dose of TCAE at first and then combined with amphotericin B (AmB) of HT-29. AmB is a common antifungal agent used to treat systemic fungal infection by direct binding to ergosterol resulted in producing pore in fungal membrane which led to cell death. Therefore, we hypothesized that the extract of T. camphoratus contains a certain kind of sterol that has higher affinity to AmB and these sterols can easily replace the existing cholesterol in the cell membrane of cancer cells and then the replaced sterols became the target of AmB later on. The hypotheses also suggested the the phospholipid composition of cancer cells might be different from normal cells that attracted easier than normal cells to the sterols from T. camphoratus. Organic solvents in different polarity were employed to extract solid state cultivated T. camphoratus and n-hexane was found to be the most effective solvent for the extraction of AmB synergistic component to inhibit the growth HT-29 cell line other than TCAE. The n-hexane extract was separated by preparative thin layer chromatography (PLC) and two fractions (SB and LR) were found to be active in synergistic to AmB. SB demonstrated blue band under 254 nm UV and LR was a red color fraction under visible light spectrum with the yield around 0.01% for both fractions. MTT assay indicated that LR fraction had a low cytotoxicity to both HT-29 and normal cell HEK-293 when LR and SB is used alone with the relative survival rate at 80.87 % and 64.12 %, respectively. However, if 5μg/ml of LR was pretreated to HT-29 and combined with 3 μg/ml of AmB for 24 hr later, the survival rate was dropped to 47.3±3.88 %. Three components were obtained LR-LG, LR-1 and LR-R by further purification by PLC. LR-LG and LR-R were suggested to be the target components with a synergistic effect in dose dependent manner with AmB. The combined use of LR-LG / LR-R and AmB could be useful for the therapy of cancer for both the doses of LR-LG/LR-R and AmB can be minimized and less cytotoxicity was observed to normal cell in this administration system. The chemical structure of LR-LG and LR-R are under investigation.

主目錄
致謝 I
中文摘要 II
英文摘要 IV
主目錄 1
圖表目錄 3
第一章 研究內容 5
1-1 前言 5
1-2 文獻回顧 8
1-2-1 細胞膜之組成 8
1-2-2 脂質 9
1-2-3 牛樟芝 11
1-2-3-1 命名與分類 12
1-2-3-2 生理活性 13
1-2-3-3 多醣體 14
1-2-3-4 三萜類 15
1-2-3-5 抗癌功能 19
1-2-4 兩性黴素B 29
1-2-4-1 作用與用途 29
1-2-4-2 藥理機制 29
1-2-4-3藥物用量及其副作用 29
1-2-4-4 討論 30
第二章 研究目標 33
第三章 實驗材料與方法 34
3-1 細胞來源與培養條件 34
3-1-1 細胞來源 34
3-1-2培養條件 34
3-2 實驗材料與儀器 35
3-3 實驗流程圖 38
3-4 實驗方法 39
3-4-1 三萜類萃取及純化分析 39
3-4-1-1 樟芝有效成分之萃取 39
3-4-1-2 TLC薄層液相色層分析法 39
3-4-1-3 PLC 製備型液相色層分析法 40
3-4-1-4 HPLC 高效能液相色層分析法 40
3-4-2 樟芝對於大腸癌細胞株與正常細胞株之影響 41
3-4-2-1 四錯鹽分析法測試細胞毒性(MTT tetrazolium salt assay)： 41
3-4-2-2 細胞膜脂質萃取 42
3-4-2-3 統計分析 42
第四章 實驗結果 43
4-1 樟芝與兩性黴素B結合能力 43
4-2 樟芝與兩性黴素B不同濃度對於大腸直腸癌細胞株(HT-29)與正常細胞株(HEK-293)影響 46
4-3 樟芝與兩性黴素B結合能力對於大腸直腸癌細胞株(HT-29)的影響 49
4-4 樟芝不同極性萃取方式對於大腸直腸癌細胞株(HT-29)的影響 50
4-5 正己烷(n-Hexane)萃取之化合物對於大腸直腸癌細胞株(HT-29)的影響 51
4-6 正己烷(n-Hexane)萃取之LR分析 54
4-7 正己烷(n-Hexane)萃取之LR對於大腸直腸癌細胞株(HT-29)的影響 58
4-8 酒精萃取之樟芝粗萃物對於大腸直腸癌細胞株(HT-29)與正常細胞株(HEK-293)細胞膜脂質的影響 60
第五章 實驗討論 61
第六章 結論 64
第七章 參考文獻 66

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