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    請使用永久網址來引用或連結此文件: http://ir.cmu.edu.tw/ir/handle/310903500/41247


    題名: 血藤莖部粗抽物誘導人類血癌細胞凋亡並透過活性氧化物途徑與三氧化二砷產生協同效果之抗血癌作用
    Synergistic Apoptosis-Inducing Antileukemic Effects of Mucuna macrocarpa Stem Extract and Arsenic Trioxide in Human Leukemic Cells via a Reactive Oxygen Species-Dependent Mechanism
    作者: 盧冠宏
    貢獻者: 中國藥學暨中藥資源學系博士班
    關鍵詞: 血藤;白血病;細胞凋亡;三氧化二砷;活性氧化物 Mucuna macrocarpa;leukemia;apoptosis;arsenic trioxide;ROS
    日期: 2011-07-27
    上傳時間: 2011-10-17 16:00:20 (UTC+8)
    出版者: 中國醫藥大學
    摘要: 血藤 (Mucuna macrocarpa Wallich) 為豆科血藤屬植物，遍布於台灣及東南亞中低海拔山區，民間以其莖部入藥，治療身體循環不佳或是血液相關疾病，屬活血化瘀劑。本研究的目的在於探討血藤是否具有抗血癌的功效，以其驗證民俗用法並提升應用價值。首先，利用體外細胞培養模式測試血藤莖部粗抽物 (CMEMM) 對於人類血癌細胞如HL-60, Jurkat和Molt-3的影響。實驗結果顯示，劑量範圍在25 g/ml至75 g/ml之CMEMM對於HL-60細胞有抑制生長的效果，且有時間及劑量的依存關係，其72小時之半數抑制濃度 (IC50) 為36.4 μg/ml。而CMEMM誘導HL-60細胞凋亡的現象可經由細胞核凋亡性形態特徵的出現、細胞處於sub-G1期比率的增加、annexin V-positive比率的提升，以及細胞內活化態caspase-3的提升等結果證實。此外，CMEMM於活體動物模式中亦有顯著抑制腫瘤生長的效果，且實驗過程中對於老鼠不具毒性。
    為拓展臨床上的應用價值，本研究進一步評估CMEMM與血癌治療用藥三氧化二砷 (ATO) 並用的治療效果。實驗結果發現，劑量為50 μg/ml之CMEMM 與2.5 μM之ATO並用時可協同性地抑制HL-60和Jurkat細胞生長，而CMEMM與ATO並用所誘導的細胞凋亡，亦由增加細胞處於sub-G1期的比率、提升annexin V-positive的比率，以及出現細胞核凋亡性形態特徵等結果得到驗證，且發現此現象可能與粒線體路徑中caspase-3和caspase-9等細胞凋亡相關性蛋白質被活化所致。此外，CMEMM與ATO並用會提高細胞內活性氧化物的生成，而當抗氧化劑如N-acetyl cysteine (NAC), butylated hydroxytoluene或α-tocopherol介入時，此細胞凋亡現象將顯著地受到抑制，其機制與抗氧化劑如NAC會抑制粒線體路徑中caspase-3和caspase-9等蛋白質有關。
    綜合上述結果，本論文證實台灣藥用植物血藤之抗血癌活性及其機制，並以合併使用血癌治療用藥三氧化二砷的效果，提供未來臨床研究的基礎，亦開啟血癌治療上以中草藥與化療藥物並用來提升治療效果的可能性。
    Mucuna macrocarpa Wallich (Leguminosae) is believed to hold blood circulation activating effects, and has been used as a folk remedy in Taiwan and Southeast Asia for the treatment of various hematologic and circulatory related ailments. The objective of this study was to investigate whether crude methanolic extract of M. macrocarpa (CMEMM) possessed antileukemic effects on human leukemia cell lines such as HL-60, Jurkat and Molt-3. CMEMM was prepared from dried stems of this plant, and its apoptosis-inducing effects were investigated using HL-60 cells in vitro and in vivo first. With treatment of 25 to 75 g/ml CMEMM, the in vitro antiproliferative effect on HL-60 cells increased in a concentration- and time-dependent manner during the 72 h treatment period. The concentration of CMEMM that exhibited a 50% growth inhibition (IC50) for 72 h exposure was 36.4 μg/ml. Apoptosis triggered by CMEMM in HL-60 cells was confirmed by characteristic apoptotic nuclear fragmentation, concentration-dependent accumulation of sub-G1 phase in cell cycle analyses, increased percentages of annexin V-positive apoptotic cells, and concentration-dependent elevation of active caspase-3. Furthermore, the in vivo effect of tumor growth suppression by CMEMM (500 mg/kg/day i.p.) was observed in mouse xenografts.
    To broaden the therapeutic potential of CMEMM, further study was to examine the potential of enhancing the antileukemic activity of arsenic trioxide (ATO) using combination treatment. Human leukemia cells HL-60, Jurkat and Molt-3 were treated with various doses of CMEMM, ATO and combinations thereof for 24 and 48 h. Results indicated the combination of 50 μg/ml CMEMM and 2.5 μM ATO synergistically inhibited cell proliferation in HL-60 and Jurkat cells. Apoptosis triggered by CMEMM/ATO treatment was confirmed by accumulation of cells in the sub-G1 phase in cell cycle analyses, characteristic apoptotic nuclear fragmentation and increased percentage of annexin V-positive apoptotic cells. Such combination treatments also led to elevation of reactive oxygen species (ROS). The anti-oxidants N-acetyl cysteine (NAC), butylated hydroxytoluene, and α-tocopherol prevented cells from CMEMM/ATO-induced apoptosis. The CMEMM/ATO-induced activation of caspase-3 and -9 can be blocked by NAC.

    In conclusion, these results suggest that CMEMM/ATO-combination treatment exerts synergistic apoptosis-inducing effects in human leukemic cells through a ROS-dependent mechanism and may provide a promising antileukemic approach in the future.
    顯示於類別:[中國藥學暨中藥資源學系暨碩博班] 博碩士論文

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