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研究生(外文):Shao-Cheng Wang
論文名稱(外文):The influence of oligomeric polyphenolics from tea and longan flower on growth in human colorectal carcinoma cell lines
指導教授(外文):Chih-Cheng LinChih-Ping Hsu
外文關鍵詞:cell cyclecolorectal carcinomalongan floweroligomeric polyphenolicstea
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許多研究報告指出,攝取富含多酚類化合物的天然食材具有抗氧化活性,可降低癌症與心血管疾病等疾病的發生率。本研究探討龍眼花與茶水萃出物中富含的寡聚合茶多酚對人類大腸直腸癌細胞株生長的抑制能力與作用機制,實驗中以生長特性貼附型的SW480與半懸浮型的Colo 320DM當做實驗細胞株,觀察寡聚合多酚對其細胞生長抑制、細胞週期、細胞內粒線體膜電位及誘導細胞凋亡作用的變化。為探討不同聚合度多酚將茶抽出物(TMT)以膜分離技術將茶多酚區分為中度聚合多酚(MMT)與高度聚合多酚(HMT)與龍眼花(LFE)進行比較。實驗結果顯示這些試樣均具有相當於(-)-Epigallocatechin gallate(EGCG)等化合物的抗氧化活性。在大腸直腸癌細胞凋亡的結果中發現以龍眼花抑制能力最佳,而茶多酚中以中度聚合茶多酚效果最好;在處理48小時後,MMT、HMT、TMT及LFE對SW480生長抑制的IC50分別為302.41、336.85、407.24及151.76 μg/mL,對Colo 320DM則為177.06、197.95、224.18及111.73 μg/mL。由細胞週期分析的結果可知:樣品濃度在200 μg/mL時,能使兩株大腸直腸癌細胞細胞週期停滯在代表DNA複製的S phase。這個S phase的停滯作用與細胞內細胞週期調控蛋白Cyclin A下降和Cyclin E上升有關。在偵測細胞內粒線體膜電位的實驗結果發現:隨著萃出物添加濃度的增高,其粒線體膜電位的下降情形亦有增加,顯示添加多酚會使大腸直腸癌細胞凋亡的原因與粒線體膜損傷有關。此外細胞凋亡原因與Caspase 3的活化有關,且Colo 320DM較SW480明顯。本研究證明,寡聚合多酚將細胞週期停滯在S phase而抑制腫瘤細胞生長的能力,亦會有導致粒線體膜電位下降及誘導細胞凋亡的能力。
Previous research is proved that absorption of consumption polyphenolic-rich subsidiaries can decrease the incidence of diseases, such as cancer and coronary artery disease. The aim of this thesis was to investigate the influence of oligomeric polyphenolics from tea and longan flower on the growth of human colorectal carcinoma cell lines(SW480 and Colo 320DM). The tea was fractionated to oligomer-rich and polymer-rich extract by membrane separation. Cell cycle analysis, mitochondria membrane potential, membrane alteration and protein expression were determined in this thesis. All the extracts contained polyphenolic compounds and showed prominent antioxidant activity. The results showed that oligomeric polyphenolics, especially longan flower, systemically inhibited the proliferation of SW480 and Colo 320DM in a dose dependent manner. Longan flower tea extract showed strong S phase blocking activity at the concentration of 200 μg/mL to 400 μg/mL on these two cell lines. Protein expression results showed the close relationship between S phase arrest and cell cycle regulators cyclin A and cyclin E. The mitochondria membrane potential of treated cells was also loss. Longan flower extract increased apoptoic cell death at the concentration of 400 μg/mL on SW480 and 200 μg/mL on Colo 320DM. According to these results revealed that oligomeric polyphenol have anti-proliferative, S phase blocking, mitochondria membrane potential loss and apoptosis inducing activity. These results provide a further evaluation for its application as a nature cancer chemopreventive agent and health food.
目 錄
誌謝 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 VI
表目錄 VII
第一章 緒論 1
1.1 前言 1
1.2 茶葉之簡介 2
1.3 龍眼花之簡介 9
1.4 氧化壓力與疾病 13
1.5 大腸直腸癌 15
1.6 細胞週期(Cell cycle) 20
1.7 細胞凋亡(Apoptosis) 22
1.8 研究目的與動機 27
第二章 研究材料與方法 28
2.0 實驗架構流程圖 28
2.1 實驗樣品 29
2.1.1 樣品來源 29
2.1.2 樣品之萃取製備 29
2.2 實驗細胞株 29
2.3 實驗藥品與試劑 30
2.4 常用儀器設備 32
2.5 實驗方法 33
2.5.1 成分分析 33總酚類化合物(Total polyphenol) 33總類黃酮(Total flavonoids) 33縮合型單寧(Condensed tannin) 33原花青素(Proanthocyanidin) 33
2.5.2 抗氧化活性檢測 34 DPPH自由基清除能力 34總抗氧化能力(TEAC) 35 還原能力 35
2.5.3 細胞培養 36人類大腸癌細胞SW480、Colo 320DM繼代培養 36 細胞凍存 36 細胞解凍 36 細胞存活率分析(Trypan blue dye exclusion assay) 36 細胞週期分析(Cell cycle analysis) 37 細胞內粒線體膜電位(ΔΨm)分析 37 細胞凋亡分析(Annexin V-FITC) 38
2.5.4 西方墨點法(Western blot) 38 試劑 38 蛋白質萃取及定量 40 蛋白質電泳及轉印 40 免疫墨點法 40
2.6 統計分析 41
第三章 結果與討論 42
3.1 成分分析 42
3.2 抗氧化活性分析 44
3.3生長抑制及相關分子機制 50
第四章 結論 62
第五章 參考文獻 63

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