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研究生:盧柏廷
研究生(外文):Bo-Ting Lu
論文名稱:楓香葉萃取物對人類肺癌細胞A549之凋亡研究
論文名稱(外文):Effect of the extract from the leave of Liquidambar formosana induce apoptosis of human lung cancer A549
指導教授:蘇文達蘇文達引用關係
指導教授(外文):Wen-Ta Su
口試委員:劉裕國簡良榮魏暘蘇文達
口試日期:2018-07-27
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:化學工程與生物科技系化學工程碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:66
中文關鍵詞:細胞凋亡肺癌三萜類管柱層析台灣楓香
外文關鍵詞:apoptosislung cancerTriterpenesColumn chromatographyLiquidambar formosana
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楓香是一種藥物價值極高的植物,具有使癌細胞凋亡的效果。本實驗分離出楓香葉中的有效物質,並對A549/H460/HCT116/Sk-Hep-1做抗癌測試。使用氯仿將楓香葉經由微波的方式萃取,微波萃取相較於傳統熱回流萃取法更能夠縮短萃取時間並達到更高的產量,在400W, 40min, 60℃參數下得產率4.90%。再藉由正己烷、丁醇、乙酸乙酯、甲醇水溶液、氯仿不同溶劑下進行固液分離,選擇出對正常細胞L929傷害較低的正己烷萃取物繼續純化。利用管柱層析將正己烷粗萃物分離濃縮成Fraction1~Fraction12。發現Fr.10對A549的抗癌效果最高,Fr.10的IC50達119.73µg/mL,Fr.10相對於其他Fraction有較高效果,因此選擇Fr.10作為樣品進行後續實驗。
Cell Cycle觀察到SubG1凋亡峰的增加、在細胞雙染法檢測細胞活力的實驗觀察到凋亡的細胞比例隨藥物濃度逐漸上升。利用qRT-PCR檢測凋亡相關基因的相對表現量,並透過免疫螢光染色法和Western blot觀察凋亡相關蛋白的表現量變化,在由GC-MS鑑定Fr.10的活性成分為3,5-豆甾二烯(Stigmastan-3,5-diene)、γ-谷固醇(gamma-Sitosterol)。因此,實驗結果顯示,楓香葉藉由微波萃取方式使A549肺癌細胞造成凋亡,其中分離出的活性物質,未來在抗癌藥物上有很大的潛力。
In this experiment, effective substances in the leaves of Liquidambar formosana were separated, and A549/H460/HCT116/Sk-Hep-1 were tested for anticancer activity. Microwave-assisted extraction was used to extract leaves of Liquidambar formosana with chloroform, microwave-assisted extraction can shorten the extraction time and achieve higher yield than the traditional heat reflux extraction method. Yield achieved 4.90% at 400 W.40 min. 60 °C. The solid-liquid separation was carried out by different solvents of n-hexane, butanol, ethyl acetate, aqueous methanol, and chloroform. The n-hexane extraction with lower damage to L929 cell lines was selected for further purification. Column chromatography was used to separate and concentrate the n-hexane crude extract into Fraction1 to Fraction12. The anticancer effect of Fr.10 on A549 was found to be highest. The IC50 of Fr.10 was 119.73 μg/mL which means Fr.10 had a better effect than other fraction. Therefore, Fr.10 is used as a sample for follow-up experiments.
  An increase in the apoptotic peak of SubG1 was observed in Cell Cycle assay, and the proportion of apoptotic cells was gradually increased with the dose-dependent in a cell double staining assay. The relative expression of apoptosis-related genes was detected by qRT-PCR. The expression of apoptosis-related protein detected by immunofluorescence staining and western blot identified results by GC-MC confirmed that Fr.10 active components were Stigmastan-3,5-diene and gamma-Sitosterol. According to the results, the extracts of leaves of Liquidambar formosana by microwave-assisted caused apoptosis to A549 cell lines. The active substances isolated from plant have great potential for anticancer drugs in the future.
摘 要 i
ABSTRACT iii
致謝 v
目錄 vi
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1前言 1
1.2動機 2
第二章 文獻回顧 3
2.1癌症 3
2.1.1癌細胞特徵 3
2.1.2肺癌 4
2.2楓香(Liquidambar formosana Hance) 4
2.2.1起源及特徵 4
2.2.2傳統中藥材用途 5
2.2.3生物活性(bioactivity) 5
2.3萃取(extraction) 6
2.3.1靜置萃取 6
2.3.2超音波萃取 6
2.3.3超臨界萃取 6
2.3.4微波萃取 7
2.4純化分離 7
2.4.1薄膜層析法(TLC) 7
2.4.1.1 Rf值 8
2.4.1.2顯色法 9
2.4.2管柱層析法(Column chromatography) 10
2.5鑑定 11
2.5.1 GC-MS(氣相色譜質譜) 11
2.6細胞檢測技術 11
2.6.1細胞存活率分析(MTT assay) 11
2.6.2細胞週期 12
2.6.3細胞凋亡(apoptosis) 13
第三章 材料與方法 16
3.1 實驗材料/藥品/儀器 16
3.1.1 植物 16
3.1.2 動物細胞 16
3.1.3材料與藥品 16
3.1.4 儀器 18
3.2實驗流程圖 20
3.3 實驗方法 23
3.3.1微波輔助萃取 23
3.3.2 固液分離 23
3.3.3薄膜層析(TLC) 24
3.3.4管柱層析 24
3.3.5 總三萜類含量分析(比色法) 25
3.3.7 細胞培養與繼代 25
3.3.8 細胞存活率分析 (MTT assay) 26
3.3.9 Two-step cell cycle 26
3.3.10 Vitality 26
3.3.11 qRT-PCR 26
3.3.12 免疫螢光染色(Immunocytochemistry) 28
3.3.13西方墨點法(Western blot) 29
第四章 結果與討論 32
4.1最佳萃取法 32
4.2微波萃取最適化 32
4.3固液分離 36
4.4分離相篩選 37
4.5細胞篩選 39
4.6薄膜層析(TLC) 39
4.7管柱層析 40
4.8細胞存活率分析 (MTT assay) 44
4.9 GC-MS 45
4.10 細胞週期分析 (Two-step cell cycle assay) 49
4.11 細胞活力分析 (Vitality assay) 51
4.12即時聚合酶鏈鎖反應 (qRT – PCR) 53
4.13免疫螢光染色 (Immunofluorescence staining) 55
4.14西方墨點法(Western blot) 57
第五章 結論與未來發展 59
第六章 參考文獻 61
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