鯽魚膽(Pluchea indica (L.) Less)在先前的研究發現具備抗腫瘤活性也有抗潰瘍、抗發炎、抗氧化和保護肝臟的效能。目前用來選擇有效抗腫瘤藥物的重要指標，包括能否遏止細胞週期、誘導細胞凋亡與細胞分化。本研究則利用人類血癌細胞K562，評估鯽魚膽是否具有抗腫瘤的活性。利用各種不同濃度在同一時間點的處理下，觀察細胞增殖率的作用，以流式細胞儀分析細胞週期的影響及細胞凋亡的調節。鯽魚膽對人類血癌細胞K562細胞生長增殖作用，以WST-1、Alamar blue分析及Trypan blue染色計數，經不同濃度鯽魚膽處理人類血癌細胞K562，有抑制細胞增殖率的作用，且其抑制作用呈現劑量依賴性(dose-dependent)。以流式細胞儀分析經鯽魚膽處理之人類血癌細胞K562，結果顯示細胞週期停滯在G2/M期。進一步探討細胞週期相關蛋白質和細胞週期抑制者的變化。本研究亦發現鯽魚膽誘導凋亡使得細胞凋亡蛋白增加，且抗凋亡蛋白降低，造成Cytochrome C釋出以及caspase-3的活化。綜合以上結果顯示鯽魚膽抑制腫瘤細胞增殖作用主要是使細胞週期停滯在G2/M期並有誘導凋亡的現象，所以鯽魚膽可能是一種具有潛力抑制人類血癌細胞的草藥。

Previous studies have shown that Pluchea indica is a plant with anti-tumor activities, anti-ulcer, anti-inflammatory, anti-oxidant and liver-protection effects. Important index for choosing effective anti-tumor drug includes whether the cell cycle is arrested and cell apoptosis as well as cell differentiation are induced or not. This study utilized human leukemia cells K562 to evaluate whether Pluchea indica has anti-tumor activity or not. Human leukemia cells K562 were treated with different concentration of Pluchea indica extract at the same time point, then cell proliferation rate was observed, and the cell cycle effect and apoptosis regulation were analyzed by flow cytometer. WST-1, Alamar blue analysis and trypan blue stain counting were used for evaluating the effect of Pluchea indica on human leukemia cells K562 proliferation. Human leukemia cells K562 were treated with different concentrations of Pluchea indica, results were shown out that Pluchea indica could inhibit cell proliferation and the effect was dose-dependent.The treated human leukemia cells K562 treated with Pluchea indica extract were further analyzed by flow cytometry. Results suggested that cell cycle was arrested at G2/M phase, and the cell cycle and relevant protein change were further investigated. Cell cycle inhibitor were increased. This study also found that Pluchea indica extract induced apoptosis and increased cell apoptosis protein , decreased anti-apoptosis , resulting in the release of cytochrome c and activating caspase-3. In conclusion, the results demonstrated that Pluchea indica inhibits tumor cell proliferation, and induces cell cycle arrests at G2/M phase and lends to apoptosis. Therefore, Pluchea indica extract may be a potential Chinese herbal medicine to inhibit human leukemia cells.

論文審定書………………………………………………………….....…i
誌謝………………………………………………………………….....…ii
中文摘要……………………………………………………………........iii
英文摘要………………………………………..……………………......iv
目錄……………………………………………………………………....vi
第一章 緒言………………………………………………………..........1
1.1鯽魚膽(Pluchea indica (L.) Less)…………………………............1
1.2血球生成………………………………………………………..........1
1.3血癌-白血病…………………………………………………….........2
1.4人類血癌細胞株K562………………………………………............3
1.5細胞週期與調控分子………………………………………….........4
1.6細胞凋亡……………………………………………………….........6
1.7細胞凋亡之檢測方法………………………………………….........9
1.7.1細胞型態學檢測……………………………………………..........9
1.7.2流式細胞儀(flow cytometry)…………………………................10
第二章 材料與方法……………………………………………............11
2.1實驗材料……………………………………………………............12
2.1.1化學試劑………………………………………………….............12
2.1.2物品材料………………………………………………….............13
2.1.3抗體……………………………………………………….............13
2.1.4設備儀器………………………………………………….............14
2.2實驗方法…………………………………………………….............14
2.2.1鯽魚膽有機水萃分離……………………………………..............14
2.2.2鯽魚膽有機水萃物樣品配製……………………………..............15
2.2.3細胞培養…………………………………………………..............15
2.2.4細胞冷凍…………………………………………………..............15
2.2.5細胞解凍…………………………………………………..............15
2.2.6細胞增殖率(cell proliferation)分析……………………...............16
2.2.6.1 WST-1分析…………………………………………….............16
2.2.6.2細胞計數-直接顯微鏡觀察…………………………….............16
2.2.6.3細胞計數-Trypan blue染色法………………………................16
2.2.6.4 Alamar blue分析……………………………………................17
2.2.7細胞週期(cell cycle)……………………………………...............17
2.2.8 DAPI染色分析……………………………………………............17
2.2.9西方墨點法………………………………………………..............18
2.2.9.1蛋白質定量………………………………………………...........18
2.2.9.2電泳、轉漬、免疫呈色…………………………………...........18
2.2.10 Annexin V/PI 流式細胞儀分析………………………...............19
2.2.11統計分析…………………………………………………............19
第三章 結果……………………………………………….....................20
3.1 Pluchea indica會抑制人類血癌細胞K562生長…………….........20
3.2 Pluchea indica對人類血癌細胞K562細胞週期之影響…….........20
3.3 Pluchea indica調節人類血癌細胞K562細胞週期相關蛋白表現..21
3.4 Pluchea indica對人類血癌細胞K562細胞凋亡之影響…….........21
3.4.1 Annexin V/PI流式細胞儀分析……………………….................21
3.4.2 DAPI染色分析…………………………………………...............22
3.5 Pluchea indica會引起人類血癌細胞K562 cytochrome c的變化.22
3.6 Pluchea indica調節人類血癌細胞K562之相關蛋白表現.............22
第四章 討論………………………………………………………….......24
參考文獻……………………………………………………….…….......27
圖表目錄………………………………………………………………....37
附錄……………………………………………………………………....60

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