臺灣博碩士論文加值系統

大腸癌已躍昇台灣癌症發生第一位，預防與治療策略須更積極研究。芒果核仁富含多酚物質，初步證實具抑制乳癌細胞生長與引發凋零效應。本論文廣泛深入研究芒果核仁萃取物對大腸癌細胞生長是否有抑制作用，並了解其作用之細胞與分子機制。論文首先建立定溫烘乾與酒精浸泡取得芒果核仁萃取物，經過總多酚、總類黃酮、縮合單寧含量測定及清除自由基能力試驗後，以之處理正常腸壁細胞IEC-6或兩株大腸癌細胞SW480與Colo 320DM，再利用錐蟲藍染色計算細胞存活率，或染上propidium iodide再配合流式細胞儀測定細胞週期變化，或以Annexin V-FITC染出細胞凋零現象，再以Rhodamine 123染色確定粒線體膜電位是否參與細胞凋零，最後以西方墨點法驗證細胞變化的分子機制。結果發現，以定溫烘乾酒精浸泡的芒果核仁萃取物含有高量的多酚物質，但這些多酚物質僅有少量類黃酮與縮合單寧。清除DPPH自由基的能力與等量的兒茶素和鞣花酸相當。對正常腸壁細胞在50μg/mL濃度以下都沒有明顯毒性，然而對兩株試驗的大腸癌細胞展現劑量與時間相關性的生長抑制作用。細胞週期試驗確定芒果核仁萃取物抑制SW480細胞在G1期，與cyclin D1及cyclin E被抑制有關。Colo 320DM被抑制在S晚期，與cyclin B下降有關。細胞凋零試驗顯示芒果核仁萃取物對兩個細胞引發少量的粒腺體媒介的細胞凋零，這個細胞凋零可能與外在路徑活化caspase 3和PARP裂解引起的凋零反應有關。綜合以上結果，芒果核仁萃取物有較多量的多酚物質和類似兒茶素與鞣花酸的自由基清除能力，對正常細胞沒有毒性的濃度下，可以抑制至少兩株大腸癌細胞的生長，機制是透過引發細胞週期停滯與細胞凋零，芒果核仁萃取物是具有潛力的大腸癌預防與治療的物質，值得進一步深入開發。

Colorectal cancer (CRC) has become the most common cancer type in Taiwan since the dilatory behavior of Taiwan’s people is more like that of Westerners. The strategies for CRC prevention and treatment are needed to develop. Polyphenol-rich Mango (Mangifera indica) kernel extract (MSP) has been shown to inhibit the proliferation and induce apoptosis on breast cancer cells. However, the effect of MSP on colorectal cancer has not yet been investigated. This thesis investigated the effect of MSP on two colorectal cancer cell lines, SW480 and Colo 320DM, and the possible mechanisms involved. In this thesis, MSP were obtained by using ethanol extraction from heat dried-kernel powder. The phenolic compositions of the extract named total phenolics, total flavonoids and condensed tannins were determined by colorimetry. MSP-treated cells were assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for increased apoptosis by annexin V labeling, for loss of mitochondrial membrane potential by Rhodamine 123 staining, and for changes in the levels of proteins involved in cell cycle control and apoptosis by immunoblotting. MSP (12.5 ~ 50 μg/mL) could inhibit proliferation in a dose- and time-dependent manner. The cell cycle of both MSP-treated cell lines showed obvious G1 (SW480) or late S (Colo 320DM) phase block. Western blotting further showed the cell cycle block in these two cell lines was mainly due to the expression of associated cyclins. MSP treatment increased annexin V-positive and rhodamine 123-negative cells in both cells. Increased levels of the apoptosis activation protein, caspase 3, were also found in both CRC cells. The caspase 3 activation in both CRC cells by MSP was mediated by caspase 8 activation. The inhibitory effect of MSP treatment was associated with S phase block of the cell cycle and caspase 8- and mitochondria-mediated apoptosis. The results of this thesis indicate that MSP has potential to be developed as a novel chemo-preventive or chemotherapy agent for colorectal cancer.

目錄
第一章 緒論……………………………………………….. 1
1.1 大腸癌…………………………………………….. 1
1.2 芒果核仁………………………………………….. 5
1.2.1 芒果發展史……………………………………….. 5
1.2.2 芒果的藥用價值………………………………….. 7
1.3 芒果核仁抗大腸癌之可能性…………………….. 9
1.3.1 有記載的芒果藥效……………………………….. 9
1.3.2 芒果抗癌的文獻回顧…………………………….. 9
1.4 細胞週期與大腸癌……………………………….. 11
1.5 細胞凋零與大腸癌……………………………….. 12
1.6 論文研究方向…………………………………….. 13
第二章 研究架構、實驗材料與方法……………………... 15
2.1 實驗樣本………………………………………….. 15
2.2 芒果核仁之萃取………………………………….. 15
2.3 萃取方法………………………………………….. 15
2.4 成份分析………………………………………….. 16
2.4.1 總酚類化合物(Total polyphenol)含量測定…… 16
2.4.2 總類黃酮(Total flavonoid)含量測定……………16
2.4.3 縮合型單寧(Condensed tannins)含量測定………17
2.5 DPPH自由基清除能力…………………………… 17
2.6 細胞特性實驗……………………………………... 18
2.6.1 實驗用人類大腸癌細胞株………………………... 18
2.6.2 細胞存活率分析 …………………………………. 18
2.6.3 細胞週期分析……………………………………... 19
2.6.4 細胞凋零試驗……………………………………... 20
2.6.5 粒線體膜電位分析………………………………... 21
2.7 西方墨點法………………………………………... 21
2.7.1 細胞蛋白萃取……………………………………... 21
2.7.2 蛋白質濃度測定…………………………………... 22
2.7.3 SDS-聚丙烯醯胺膠體電泳……………………….. 22
2.7.4 蛋白質轉漬……………………………………….. 23
2.7.5 免疫墨點法……………………………………….. 23
2.8 統計分析………………………………………… 23
第三章 結果分析………………………………………….. 24
3.1 芒果核仁多酚(MSP)的多酚種類分析分.duvu…析… 24
3.2 MSP的抗自由基活性…………………………….. 24
3.3 MSP對正常腸壁細胞的毒性試驗……………….. 25
3.4 MSP抑制大腸癌細胞株之生長………………….. 25
3.5 MSP抑制細胞週期……………………………….. 26
3.6 MSP對大腸癌細胞週期相關蛋白之影響……….. 26
3.7 MSP對大腸癌細胞凋零之分析………………….. 27
3.8 MSP引導大腸癌細胞粒腺體膜電位喪失………... 28
3.9 MSP對大腸癌細胞凋零相關蛋白之影響……….. 29
第四章 討論……………………………………………… 30
第五章 結論……………………………………………… 36
參考文獻 …………………………………………………… 37
圖表 …………………………………………………… 46
圖表目錄
表一、 芒果核仁酒精萃取物之酚類物質含量表 …………… 50
圖一、 MSP清除DPPH自由基能力。………………………… 51
圖二、 MSP對正常細胞IEC-6的影響。……………………… 52
圖三、 MSP對大腸癌細胞SW480的影響。…………………… 53
圖四、 MSP對大腸癌細胞Colo 320DM的影響。…………… 54
圖五、 MSP對大腸癌細胞SW480細胞週期的影響。………… 55
圖六、 MSP對大腸癌細胞Colo 320DM細胞週期的影響。… 56
圖七、 MSP對大腸癌細胞SW480細胞週期相關蛋白質的影響。57
圖八、 MSP對大腸癌細胞Colo 320DM細胞週期相關蛋白質的影響。……………………………………………………….. 58
圖九、 MSP對大腸癌細胞SW480細胞凋零的影響。……………59
圖十、 MSP對大腸癌細胞Colo 320DM細胞凋零的影響。…….60
圖十一、 MSP對大腸癌細胞SW480細胞粒線體膜電位的影響。. 61
圖十二、 MSP對大腸癌細胞Colo 320DM細胞粒線體膜電位的影響。…………………………………………………………. 62
圖十三、 MSP對大腸癌細胞SW480細胞凋零相關蛋白質的影響。63
圖十四、 MSP對大腸癌細胞Colo 320DM細胞凋零相關蛋白質的影響。……………………………………………………… 64

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