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學校名稱輔仁大學
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研究生(中)鄭俊慈
研究生(英)Chun-Tsz Cheng
論文名稱(中)血液腫瘤病人骨髓基質細胞功能之探討
論文名稱(英)Studies on the function of hematological malignant bone marrow stromal cells
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指導教授(中)曾婉芳
指導教授(英)Woan-Fang Tzeng
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畢業學年度94
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關鍵字(中)骨髓 骨髓基質細胞 血液腫瘤 化學治療 細胞激素 抗氧化能力 凋亡
關鍵字(英)hematological malignant bone marrow stromal cell cytokine
摘要(中)摘要 血液腫瘤病人依其病徵在臨床上會施予化學治療、放射治療、骨髓移植、周邊血幹細胞移植或臍帶血移植。不論是化學治療或移植前之療程對病人的骨髓造血機能都可能造成影響,為了解骨髓基質細胞在化學治療後或骨髓移植療程中受損,本研究抽取各類血液腫瘤病人不同時期的骨髓液,培養其中之骨髓基質細胞,以MTT測定法評估病人骨髓基質細胞之細胞活性、生長速度、對常見的化學治療藥物之敏感性、抗氧化能力,分泌細胞激素之能力及凋亡相關訊息分子含量,並以正常人骨髓基質細胞做為對照組。結果顯示所有未經治療之血液腫瘤病人骨髓基質細胞之細胞活性均較正常人骨髓基質細胞明顯為差,其中MDS病人之細胞活性最差,且較所有其他類別之血液腫瘤病人骨髓基質細胞活性有顯著差異。除未經治療之MM病人骨髓基質細胞生長速度與正常人骨髓基質細胞無顯著差異外,未經治療之AML, diffused large B cell lymphoma (DLBL), CML及MDS病人骨髓基質細胞生長速度均較正常人骨髓基質細胞為慢且有顯著差異。所有未經治療之血液腫瘤病人骨髓基質細胞之GSH含量與DT-diaphorase, gltathione S-transferase (GST), catalase, glutathion peroxidase (GP)及superoxide dismutase (SOD)等酵素之活性均較正常人骨髓基質細胞明顯為差。所有未經治療之血液腫瘤病人骨髓基質細胞對維他命K3之敏感性均較正常人骨髓基質細胞為高且有顯著差異,其中MDS病人骨髓基質細胞對維他命K3最為敏感。未經治療之未經治療之AML, DLBL, CML及MDS病人骨髓基質細胞對Ara-C, daunomycin, doxorubicin, cyclophosphamide及vincristine均明顯較正常人骨髓基質細胞敏感。除未經治療之MM及MDS病人骨髓液內及骨髓基質細胞分泌之IL-6含量較正常人之含量明顯為高,及未經治療之MDS病人骨髓液內IL-1β之含量亦較正常人之含量明顯為高外,所有其他未經治療之血液腫瘤病人骨髓液內及骨髓基質細胞所分泌之IL-1α,IL-1β,IL-6及IL-10等之含量均較正常人之細胞激素含量明顯為低。病人骨髓基質細胞之生長速度與骨髓基質細胞之P53, PP53及Bax等蛋白之含量呈正相關,而與Bcl-2蛋白之含量呈負相關。本研究顯示,未經治療之各類血液腫瘤病人骨髓基質細胞之功能確實較正常人骨髓基質細胞功能為差,且明顯對化學治療用藥較正常人骨髓基質細胞敏感,此可能造成臨床上血液腫瘤病人在化學治療後或骨髓移植、周邊血幹細胞移植及臍帶血移植後因骨髓基質細胞受損而致造血機能不足。
摘要(英)Abstract The hematological malignant patients, based on their clinical symptoms, are treated by chemotherapy, radiotherapy, bone marrow transplantation, peripheral blood stem cell transplantation, or umbilical cord blood transplantation. Either the chemotherapy or the treatment before transplantation many affect the hematopoiesis of the patients. To investigate the damages of the bone marrow stromal cells during the chemotherapy or during the course of the transplantation treatment, we cultured the bone marrow stromal cells of the hematological malignant patients in different stages. The mitochondrial activity, the growing property, the sensitivity to drugs used in chemotherapy, the antioxidative ability, the cytokine secretion ability, and the quantity of the apoptosis-related proteins, of the bone marrow stromal cells are arrayed. Bone marrow stromal cells from normal person are used for comparison. The results indicated that the activities and the quantities of bone marrow stromal cells of the hematological malignant patients without therapy are worse than the bone marrow stromal cells from normal person in the mitochondrial activity, the quantity of GSH and the activities of antioxidated enzymes including DT-diaphorase, GST, catalase, GP and SOD, and the activities of bone marrow stromal cells of the MDS patients without therapy is the worst in all hematological malignant patients. Only the AML, DLBL, CML and MDS patients without therapy are slower than normal person in the growing property of bone marrow stromal cells. All hematological malignant patients without therapy are more sevsitive than normal person in the sensitivity to K3 of bone marrow stromal cells. MDS patients without therapy are most sevsitive in the sensitivity to K3 of bone marrow stromal cell. All hematological malignant patients without therapy are more sensitive than normal person in the sensitivity to drugs used in chemotherapy including Ara-C, daunomycin, doxorubicin, cyclophosphamide and vincristine of bone marrow stromal cells. Beside the quantity of bone marrow fluid and stromal cells secreted IL-6 of MM and MDS patients without therapy and the quantity of bone marrow fluid IL-1β of MDS patients without therapy are higher than normal person, other hematological malignant patients without therapy are lower than normal person in the quantity of bone marrow fluid and stromal cells secreted cytokine IL-1α, IL-1β, IL-6 and IL-10.The mitochondrial activity and the growing property of bone marrow stromal cells of the hematological malignant patients with the quantity of the proteins including P53, PP53 and Bax are positive correlation, but be negative correlation with the quantity of the Bcl-2 protein. The bone marrow stromal cells of the hematological malignant patients without therapy is clear worse than bone marrow stromal cells from normal person in the function, and more sensitive to drugs used in chemotherapy. The bone marrow hematopoiesis of the hematological malignant patient will be damaged during chemotherapy, bone marrow transplantation, peripheral blood stem cell transplantation, or umbilical cord blood transplantation in the clinical.
論文目次目錄 中文摘要................................................1-2 英文摘要................................................3-4 縮寫表..................................................5-6 中英文對照表.............................................7-9 緒論..................................................10-22 材料..................................................23-25 試劑配方..............................................26-27 方法 1. 骨髓基質細胞培養 (1.) 人類骨髓基質細胞培養....................................................28-29 (2.) HS-5細胞培養.........................................29 2. 保存骨髓基質細胞之方法 (1.) 人類骨髓基質細胞保存方法...............................29 (2.) 人類骨髓基質細胞解凍方法...............................29 (3.) HS-5骨髓基質細胞株保存方法.............................30 (4.) HS-5骨髓基質細胞株解凍方法.............................30 3. 細胞生長速度............................................30 4. 酵素免疫分析法 (1.) 樣品之製備............................................30 (2.) 樣品之測定 (a) 樣品IL-1α之含量測定.........................................................31 (b) 樣品IL-1β之含量測定.........................................................31 (c) 樣品IL-6之含量測定............................................................32 (d) 樣品IL-10之含量測定........................................................32 5. MTT測定法................................................32-33 6. 細胞內GSH與酵素活性之測定 (1) GSH之測定.............................................33 (2) DT-diaphorase活性之測定...............................34 (3) Glutathione S-transferase活性之測定...................34 (4) Glutathione peroxidase活性之測定......................34 (5) Superoxide dismutase活性之測定........................35 (6) Catalase活性之測定....................................35 7. 蛋白質的測定 (1) BCA蛋白質定量法.......................................35 (2) Lowry蛋白質測定法.....................................35 8. 細胞內P53, PP53, Bcl-2, Bax蛋白質含量之測定 (1) 細胞內P53蛋白質含量之測定................................36 (2) 細胞內磷酸化P53蛋白質(PP53)含量之測定.....................36 (3) 細胞內Bax蛋白質含量之測定................................37 (4) 細胞內Bcl-2蛋白質含量之測定..............................37 9. 統計分析................................................37 結果 一.骨髓基質細胞之生長及活性之評估.....................38-39 二.骨髓基質細胞對常見的化學治療藥物之敏感性............39-42 三.骨髓基質細胞抗氧化能力之評估.......................42-46 四.骨髓液內含及骨髓基質細胞分泌之細胞激素...............46-49 五.骨髓基質細胞之P53,PP53,Bcl-2與Bax等蛋白含量和細胞生長之相關性.................................................49-50 討論...............................................51-54 參考資料...........................................55-61 表................................................62-77 圖................................................78-91 附圖..............................................92-97
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