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研究生： 陳孟琦
Meng-Chi, Chen
論文名稱： 冬蟲夏草水萃液對量子點所引起之小鼠骨髓幹細胞分化抑制上的影響
Effects of Cordyceps sinensis on the inhibition of mice bone marrow cell differentiation by CdSe/ZnS quantum dots
指導教授： 江啟勳
Chi-Shiun, Chiang
口試委員:
學位類別： 碩士
Master
系所名稱： 原子科學院 - 生醫工程與環境科學系
Department of Biomedical Engineering and Environmental Sciences
論文出版年： 2007
畢業學年度： 95
語文別： 中文
論文頁數： 78
中文關鍵詞： 冬蟲夏草 、量子點 、骨髓幹細胞 、分化
外文關鍵詞： Cordyceps sinensis, quantum dots, bone marrow, differentiation
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    • 冬蟲夏草是中國人長久以來食用的中醫藥材，在許多研究中都指出冬蟲夏草有調節免疫的效果，在最近的研究中也指出冬蟲夏草水萃液有促進骨髓幹細胞分化的能力。量子點是目前備受矚目的新興螢光材料，因為其螢光表現的穩定度高，目前有許多研究運用為活細胞的追蹤劑。但近年研究發現量子點在不致細胞死亡的濃度下，對於骨髓幹細胞的標定會造成細胞分子層面上的改變，而抑制幹細胞的分化。本實驗旨在探討冬蟲夏草是否能減緩或阻止量子點對骨髓幹細胞分化的抑制作用。由本論文的實驗結果可以得知，由BMP的mRNA表現量、ALP的活性表現、鈣離子的表現量都可以看出量子點對於骨髓幹細胞的分化確實有抑制的情形；而冬蟲夏草水萃液對骨髓幹細胞的分化確實有促進的效果；結合冬蟲夏草水萃液來進行骨髓細胞的量子點標定，可以有效減緩量子點對骨髓細胞所造成分化抑制的情形。此外，我們也觀察冬蟲夏草水萃液對於活化蝕骨細胞的影響，發現冬蟲夏草水萃液能夠有效降低骨母細胞的RANKL表現量，有抑制蝕骨細胞分化成熟的潛力。

    Cordyceps sinensis is Chinese herb medicine and is frequently used as immune regulator. Recently, Cordyceps sinensis hot-water extract has also been found to have effects on the differentiation of mice bone marrow cells. With the characteristics of high fluorescence intensity and stability, quantum dots have become the most popular fluorescent tracers in living cell imagings. However, several recent studies have shown that using quantum dots to label bone marrow stem cells could inhibit their differentiation processes. For this concern, we aim to see whether the administration of Cordyceps sinensis hot-water extract can reduce the inhibitory effect of quantum dots labeling on the differentiation of bone marrow stem cell. According to our study by measuring the mRNA expression level of BMP, the activity level of ALP and the level of calcium ions, we found that quantum dots have an inhibitory effect on mice bone marrow differentiation, which can be reduced by the administration of Cordyceps sinensis. Additionally, we also found that Cordyceps sinensis hot-water extract has opposite effect on osteoclast differentiation. We found that bone marrow stem cells treated with Cordyceps sinensis hot-water extract can reduce the mRNA expression level of RANKL in osteoblasts, which consequently reduced the osteoclast differentiation.

    英文摘要…………………………………………………………………I 中文摘要………………………………………………………………III 誌謝…………………………………………………………………IV 目錄……………………………………………………………………VI 第一章 序論……………………………………………………………1 1.1冬蟲夏草……………………………………………………………1 1.1.1 簡介…………………………………………………………1 1.1.2 冬蟲夏草的功用……………………………………………2 1.2 量子點(Quantum dots) …………………………………………4 1.2.1 簡介…………………………………………………………4 1.3 骨頭分化(Osteogenesis) ………………………………………6 1.3.1 簡介………………………………………………………6 1.3.2 冬蟲夏草對骨頭分化的影響………………………………8 1.3.3 量子點對骨頭分化的影響…………………………………9 1.5 研究目的…………………………………………………………10 第二章 材料與方法……………………………………………………12 2.1 材料來源…………………………………………………………12 2.1.1 冬蟲夏草萃取液來源……………………………………12 2.1.2 冬蟲夏草的萃取…………………………………………12 2.1.3 量子點來源………………………………………………12 2.1.4 動物來源………………………………………………13 2.2 細胞培養………………………………………………………13 2.2.1 配製細胞培養液…………………………………………13 2.2.1.1 DMEM培養液……………………………………………13 2.2.1.2 α-Medium……………………………………………13 2.2.1.3 IMDM……………………………………………………14 2.2.1.4 Complete isolation edium(CIM) ……………………14 2.2.1.5 Complete expansion medium(CEM) ……………………14 2.2.2 骨髓幹細胞分化培養液配方……………………………15 2.2.3細胞繼代…………………………………………………16 2.2.4 小鼠骨髓細胞萃取………………………………………16 2.2.5 建立持續性小鼠骨髓細胞株(CIB3) ………………17 2.2.6 細胞骨頭分化表現測試………………………………18 2.3 細胞RNA表現……………………………………………………18 2.3.1 RNA的製備(Total RNA isolation) ………………………18 2.3.2 RT-PCR……………………………………………………20 2.3.3 RNA定量(Real-time PCR) ………………………………22 2.4 細胞蛋白質表現…………………………………………23 2.4.1 蛋白質粗萃取…………………………………………23 2.4.1.1 Single detergent lysis buffer的配置…………23 2.4.1.2 細胞蛋白質粗萃取……………………………………23 2.4.2 蛋白質濃度測試…………………………………………23 2.4.3 鹼性磷酸脢測試(ALP detection) ……………………24 2.5 細胞鈣離子表現…………………………………………………24 2.5.1 鈣離子染色(Alizarin red s stain) ……………………24 2.5.2 鈣離子定量……………………………………………25 2.6 細胞存活試驗(MTT assay) ……………………………………25 2.7 量子點存留試驗………………………………………………26 2.7.1 細胞流式儀分析(Flowytometry analysis) ……………26 2.7.2 螢光顯微鏡觀察…………………………………………26 第三章 實驗結果……………………………………………………27 3.1 持續性小鼠骨髓細胞株之建立…………………………………27 3.1.1 持續性小鼠骨髓細胞株的型態…………………………27 3.2 冬蟲夏草與量子點對骨頭分化之影響…………………………27 3.2.1 量子點對細胞的毒性…………………………………28 3.2.2 細胞對量子點的吞噬作用……………………………28 3.2.3 冬蟲夏草與量子點對骨頭分化的影響–細胞型態……30 3.2.4 冬蟲夏草與量子點對骨頭分化的影響–RNA表現量……31 3.2.5 冬蟲夏草與量子點對骨頭分化的影響–酵素活性……32 3.2.6 冬蟲夏草與量子點對骨頭分化的影響–鈣離子表現…34 3.3 冬蟲夏草對蝕骨細胞活性的影響………………………………35 3.3.1 RANKL mRNA的表現量…………………………………35 3.3.2 TGF-β的mRNA表現量………………………………37 第四章 討論………………………………………………………39 4.1 持續性的小鼠骨髓細胞株CIB3…………………………………39 4.2 細胞吞噬量子點的行為……………………………………39 4.3 量子點與冬蟲夏草萃取液對骨頭分化的影響…………………41 4.4 冬蟲夏草萃取液對於腫瘤轉移的潛在角色……………………45 圖表說明…………………………………………………………49 圖表………………………………………………………………56 附錄一……………………………………………………………74 參考文獻………………………………………………………………75

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