臺灣博碩士論文加值系統

香椿（Toona sinensis，TS）含有酚類及類黃酮等化合物，近年來研究發現具有降血糖及抗癌等功效。本實驗室已得知由香椿葉萃取液（TSL-1）能誘導人類急性骨髓血癌細胞（Human Premyelocytic Leukemia Cells； HL-60）之細胞凋亡，但其機制並不清楚。因此，本實驗將由細胞實驗（in vitro）和動物實驗（in vivo）兩方面來更進一步探討TSL-1對HL-60細胞造成細胞凋亡之機制為何？結果由流式細胞儀分析發現細胞週期（Cell cycle）停滯在G0/G1期。以西方墨點法分析調控細胞週期的相關蛋白質分子表現，發現 TSL-1可降低HL-60細胞之Cyclin D1、CDK4、Cyclin E、CDK2及Cyclin A蛋白質的表現，增加p15和p27蛋白質的表現。在ROS方面，TSL-1會促進HL-60細胞ROS的釋放，造成粒線體受損，導致粒線體膜電位（mitochondrial membrane potential）下降，並活化Caspase 9，進而誘導細胞凋亡。在死亡受器路徑（Death receptor pathway）發現TSL-1也活化Caspase 8，進而誘導細胞凋亡。在活體動物抗癌試驗（Xenograft tumor assays）方面，TSL-1 可有效抑制腫瘤的生長。本研究之結果為香椿具有保健食品之功效，可開發作為化學預防（chemoprevention）的保健食品及藥品。

樟芝(Antrodia cinnamomea；AC)，是一種生長在牛樟樹上的蕈類，且為台灣特有的傳統中藥。先前本實驗室已得知由樟芝菌絲發酵液(AC-10)能誘導人類急性骨髓血癌細胞(Human Premyelocytic Leukemia Cells； HL-60)之細胞凋亡，但其機制並不清楚。因此，本實驗將由細胞實驗(in vitro)和動物實驗(in vivo)兩方面來更進一步探討AC-10對HL-60細胞造成細胞凋亡之機制為何？結果由流式細胞儀分析發現細胞週期(Cell cycle)停滯在G0/G1期。以西方墨點法分析調控細胞週期的相關蛋白質分子表現，發現 AC-10可降低HL-60細胞之Cyclin D1、CDK4、Cyclin E、CDK2、及Cyclin A蛋白質的表現，增加p15和p21蛋白質的表現。在ROS方面，AC-10會促進HL-60細胞ROS的釋放，造成粒線體受損，導致粒線體膜電位(mitochondrial membrane potential)下降，並活化Caspase 9，進而誘導細胞凋亡。在死亡受器路徑(Death receptor pathway)發現AC-10也活化Caspase 8，進而誘導細胞凋亡。在活體動物抗癌試驗(Xenograft tumor assays)方面，AC-10可有效抑制腫瘤的生長。本研究之結果為樟芝具有保健食品之功效，可開發作為化學預防(chemoprevention)的保健食品及藥品。

Previous phytochemical work on Toona sinensis (TSL-1) had led to phenolic compounds and flavonoids.Plants possess many phytochemicals with various bioactivities , which include hypoglycemic effect and anticancer activities. According to the previous data, we also analyzed its cytotoxic effect on human premyelocytic leukemia HL-60 cells and underlying mechanisms. In this study, we have addressed the cytostatic and apoptosis effects of Toona sinensis (TSL-1) on human premyelocytic leukemia HL-60 cells in vitro and in vivo. Treatment of HL-60 cells with Toona sinensis (TSL-1) resulted in the inhibition of cell proliferation , arrest of cell cycle in G0/G1 phase and commitment to apoptosis. The results also demonstrated that Toona sinensis (TSL-1) down-regulate the expression of cell cycle progress factors cyclin D1, CDK4, cyclin E, CDK2 and cyclin A .The Toona sinensis (TSL-1) up-regulate the expression of cell cycle progress factors p15 and p27. Toona sinensis (TSL-1) also induced production of reactive oxygen species and decreased the levels of mitochondria membrane potential in HL-60 cells. The active form of apoptosis key enzyme caspase-8 and caspase-9 was increased. Our results revealed that treatment of Toona sinensis (TSL-1) inhibits HL-60 tumor growth in experiments. Whereas, Toona sinensis (TSL-1) did not show any side effects in these in vivo studies. Our results suggest Toona sinensis (TSL-1) that could be used as a health food preventing and alleviating leukemia proliferation and possess the potential to be developed as an anticancer drug.

Antrodia cinnamomea (AC-10) is well known in Taiwan as a traditional Chinese medicine. According to the previous data, we also analyzed its cytotoxic effect on human premyelocytic leukemia HL-60 cells and underlying mechanisms. In this study, we have addressed the cytostatic and apoptosis effects of Antrodia cinnamomea (AC-10) on human premyelocytic leukemia HL-60 cells in vitro and in vivo. Treatment of HL-60 cells with Antrodia cinnamomea (AC-10) resulted in the inhibition of cell proliferation , arrest of cell cycle in G0/G1 phase and commitment to apoptosis. The results also demonstrated that Antrodia cinnamomea (AC-10) down-regulate the expression of cell cycle progress factors cyclin D1, CDK4, cyclin E, CDK2 and cyclin A .The Toona sinensis up-regulate the expression of cell cycle progress factors p15 and p21. Antrodia cinnamomea (AC-10) also induced production of reactive oxygen species and decreased the levels of mitochondria membrane potential in HL-60 cells. The active form of apoptosis key enzyme caspase-8 and caspase-9 was increased. Our results revealed that treatment of Antrodia cinnamomea (AC-10) inhibits HL-60 tumor growth in experiments. Whereas, Antrodia cinnamomea (AC-10) did not show any side effects in these in vivo studies. Our results suggest Antrodia cinnamomea (AC-10) that could be used as a health food preventing and alleviating leukemia proliferation and possess the potential to be developed as an anticancer drug.

第一部分 香椿（TSL-1）對人類急性骨髓血癌細胞凋亡及細胞週期 的機制探討

中文摘要及英文摘要
縮寫表
第一章、文獻整理及研究動機 -------------------------------1
一、香椿之介紹 -------------------------------------------1
二、血癌 -------------------------------------------------4
二、細胞週期與癌症的關係 ---------------------------------7
四、細胞凋亡之介紹 --------------------------------------13
五、粒線體與細胞凋亡之關連性 ----------------------------18
六、活性氧化物 (reactive oxygen species；ROS) 與細胞凋亡之關連性 --------------------------------------------------20
七、裸鼠異位移植腫瘤 (nude mice xenograft tumor model) 模式 ------------------------------------------------------21
八、研究動機 --------------------------------------------23
第二章、 實驗設計架構圖 ---------------------------------25
第三章、 實驗器材 ---------------------------------------26
一、實驗材料 --------------------------------------------27
二、儀器 ------------------------------------------------29
第四章、 實驗方法 ---------------------------------------30
第一部分：探討香椿成分TSL-1對人類急性前骨髓血癌細胞株（HL-60）的抑癌影響
一、人類急性骨髓血癌細胞(HL-60)培養 ---------------------31
二．香椿(Toona sinensis；TS)之配製 ----------------------32
三、細胞存活率(viability)之測定 -------------------------33
四、以倒立式位相差顯微鏡觀察細胞型態(Morphology) --------33
五、利用流式細胞儀(Flow cytometry)探討香椿萃取液對HL-60細胞週期之影響 ----------------------------------------------33
六、利用流式細胞儀探討香椿萃取液對HL-60細胞 sub G1 phase的分布情形 ------------------------------------------------35
七、粒線體膜電位△Ψm的分析 ------------------------------36
八、活性氧化物測定(Reactive oxygen species) -------------36
九、西方墨點法(Western blotting)分析細胞蛋白質的表現 ----37
十、實驗統計 --------------------------------------------44
第二部分：探討香椿成分TSL-1在活體動物抗癌作用
一、人類急性骨髓血癌細胞(HL-60)培養 ---------------------45
二、實驗動物 --------------------------------------------46
三、裸鼠異位移植腫瘤 (nude mice xenograft tumor model)模式----------------------------------------------------------46
四、給藥之方式 ------------------------------------------47
五、活體觀察 --------------------------------------------47
六、組織包埋和切片 --------------------------------------47
七、病理 H&E 染色法 -------------------------------------48
第五章、 實驗結果與圖表 ---------------------------------50
第一部分：探討香椿成分TSL-1對人類急性前骨髓血癌細胞株（HL-60）的抑癌影響。
第一節 香椿萃取液(TSL-1)在HL-60細胞株對細胞存活的影響與細胞型態上的改變情形 --------------------------------------52
第二節 香椿萃取液(TSL-1)在HL-60細胞株對細胞週期(Cell cycle)的影響 --------------------------------------------56
第三節 測定香椿萃取液(TSL-1)在HL-60細胞株是否產生活性氧化物(Reactive oxygen species) -----------------------------59
第四節 香椿萃取液(TSL-1)在HL-60細胞株對膜電位(Mitochondrial membrane potential)的影響 ----------------62
第五節 利用西方墨點法(Western blotting)探討香椿萃取液(TSL-1)對細胞週期調控蛋白質表現量的影響 -----------------65
第六節 利用西方墨點法(Western blotting)探討香椿萃取液(TSL-1)對Casapse-9 蛋白表現量的改變 ---------------------75
第七節 利用西方墨點法(Western blotting)探討香椿萃取液(TSL-1)對死亡受器路徑中調控蛋白質表現量的影響 -----------77
第八節 利用西方墨點法(Western blotting)探討香椿萃取液(TSL-1)對SOD及Catalase蛋白質表現量的影響 ----------------82
第二部分：探討香椿成分TSL-1在活體動物抗癌作用
第一節 香椿萃取液 (TSL-1) 可抑制血癌 (HL-60) 腫瘤的生長-----------------------------------------------------------86
第六章、 討論 -------------------------------------------92
第七章、 結論 -------------------------------------------98
第八章、 參考文獻 --------------------------------------100


第二部分 樟芝（AC-10）對人類急性骨髓血癌細胞凋亡及細胞週期的機制探討

中文摘要及英文摘要
縮寫表
第一章、 文獻整理及研究動機 ----------------------------114
一、 樟芝之介紹 ------------------------------------115
二、 研究動機 --------------------------------------127
第二章、 實驗設計架構圖 --------------------------------128
第三章、 實驗器材 --------------------------------------130
一、實驗材料 -------------------------------------------131
二、儀器 -----------------------------------------------133
第四章、 實驗方法---------------------------------------134
第一部分：探討樟芝發酵液（AC-10）對人類急性前骨髓血癌細胞株（HL-60）的抑癌影響。
一、人類急性骨髓血癌細胞(HL-60)培養 --------------------135
二．樟芝(Antrodia Camphorata；AC)之配製 ----------------136
三、細胞存活率(viability)之測定 ------------------------137
四、以倒立式位相差顯微鏡觀察細胞型態(Morphology) -------137
五、利用流式細胞儀(Flow cytometry)探討樟芝發酵液對HL-60細胞週期之影響 ---------------------------------------------138
六、利用流式細胞儀探討香椿萃取液對HL-60細胞sub G1 phase的分布情形 -------------------------------------------------139
七、粒線體膜電位△Ψm的分析 -----------------------------140
八、活性氧化物測定(Reactive oxygen species) ------------140
九、西方墨點法(Western blotting)分析細胞蛋白質的表現 ---141
十、實驗統計 -------------------------------------------148
第二部分：探討樟芝發酵液（AC-10）在活體動物抗癌作用。
一、人類急性骨髓血癌細胞(HL-60)培養 --------------------149
二、實驗動物 -------------------------------------------150
三、裸鼠異位移植腫瘤 (nude mice xenograft tumor model)模式 -----------------------------------------------------150
四、給藥之方式 -----------------------------------------151
五、活體觀察 -------------------------------------------151
六、組織包埋和切片 -------------------------------------151
七、病理 H&E 染色法 ------------------------------------152
第五章、 實驗結果與圖表 --------------------------------152
第一部分：探討樟芝發酵液（AC-10）對人類急性前骨髓血癌細胞株（HL-60）的抑癌影響。
第一節、 樟芝(AC-10)在HL-60細胞株對細胞存活的影響與細胞型態上的改變情形 -------------------------------------------155
第二節、 樟芝發酵液(AC-10)在HL-60細胞株對細胞週期(Cell cycle)的影響 -------------------------------------------158
第三節、樟芝發酵液(AC-10)在HL-60細胞株是否產生活性氧化物(Reactive oxygen species) ------------------------------161
第四節、樟芝發酵液(AC-10)在HL-60細胞株對膜電位(Mitochondrial membrane potential)的影響 ---------------165
第五節 利用西方墨點法(Western blotting)探討樟芝發酵液(AC-10)對細胞週期調控蛋白質表現量的影響 --------------------168
第六節 利用西方墨點法(Western blotting)探討樟芝發酵液(AC-10)對Casapse-9 蛋白表現量的改變 ------------------------178
第七節 利用西方墨點法(Western blotting)探討樟芝發酵液(AC-10)對死亡受器路徑中調控蛋白質表現量的影響 --------------180
第八節 利用西方墨點法(Western blotting)探討樟芝發酵液(AC-10)對SOD及Catalase蛋白質表現量的影響--------------------186
第二部分：探討樟芝發酵液（AC-10）在活體動物抗癌作用。
第一節 樟芝發酵液(AC-10)可抑制血癌 (HL-60) 腫瘤的生長 -----------------------------------------------------------190
第六章、 討論 ------------------------------------------197
第七章、 結論 ------------------------------------------202
第八章、 參考文獻 --------------------------------------204

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