臺灣博碩士論文加值系統

由三氧化二砷對於治療急性髓性白血病（M3）之病人是一有效的藥劑。在一些體外實驗中發現到此藥劑對於癌症治療的作用機轉的機制主要在於停止細胞週期、造成細胞凋亡及促進分化。
而在我們的實驗主要是研究三氧化二砷對於非-M3的急性髓性白血病的治療效果。首先，先利用六至八週大的NOD/SCID老鼠建立一動物模型，以靜脈注射的方式將老鼠注射入HL-60 的細胞株以及病人白血病細胞，注射四個星期後，抽出小鼠之骨髓細胞利用單株抗體（hCD45-FITC、hCD33-PE）來作觀察，並給予小鼠注射三氧化二砷作藥物治療（5ug/0.5ml/per day），在四至六週的療期後，發現注射三氧化二砷的實驗組與注射PBS的對照組，HL-60細胞所佔之百分比並無明顯的差別。另外，進一步分析發現骨髓細胞的細胞週期發現大都停於G0/G1的階段中。而在體外培養的實驗中發現有與基質細胞直接接觸的白血病細胞比未與間質細胞直接接觸的白血病細胞所產生的自我凋亡細胞數目明顯較少。所以由此可得知，小鼠骨髓中之微環境會保護細胞避免產生自我凋亡。另外，由小鼠骨髓細胞發現細胞體內含有大量的麩胱甘（glutathion），因為體內含有轉移酶能利用體內的麩胱甘來將體內的過量的砷轉化進而代謝掉，也許這就是小鼠對於所治療的三氧化二砷沒有療效的原因之一。
由以上的結果可以知道三氧化二砷對於非急性髓性白血病（M3）並不能達到預期之療效，而造成這樣的結果有許多種原因，例如：小鼠體內的細胞停於細胞休息期（G0/G1 phase）中、小鼠體內的微環境保護白血病細胞避免三氧化二砷的毒殺、或是小鼠體內快速產生抗藥性所造成的結果。

Arsenic trioxide（ATO） is a novel agent for acute promylocytic leukemia (APL).In vitro studies have demonstrated that arsenic trioxide induces cell cycle arrest and apoptosis in multiple cancers.
We wanted to examine the effects of arsenic trioxide in non-APL acute myeloid leukemia by using a NOD/SCID mice animal model. HL-60 leukemia cell line （AML-M2） and primary cells （AML-M2）were injected into 6- to 8- week-old irradiated mice intravenously and after 4 weeks of injection, the presence of leukemic cells was verified by antibodies (hCD45-FITC、hCD33-PE). Leukemic mice were then treated with arsenic trioxide（5ug/0.5ml/per day）. After 4-6 weeks of treatment, the percentage of HL-60 cell in the bone marrow was not significantly different between control and treatment groups. Analyses of bone marrow leukemic cell revealed a high percentage cells arrested in G0/G1 phase. In vitro culture of HL-60 cells with murine BM MS-5 stromal cells also showed cell cycle arrest at G0/G1 phase. As the cytotoxicity of ATO is dependent might upon cell cycle. So the bone marrow microenvironment protects the cells from arsenic trioxide-induced apoptosis, and makes the leukemic cell insensitive to arsenic trioxide. We also found that of bone marrow leukemic cell had a higher level of intracellular glutathion, which might be the reason why the leukemic cells rapidly developed resistance to ATO.
Our study shows that ATO is not effective in non-APL in vivo, possibly by multiple mechanisms: （1）marked cell-cycle arrest in vivo, （2）protective effects by BM microenvironment from the cytotoxicity of ATO, and （3）rapid emergence of resistant cells.

第一章、 緒論…………………………………………………………… 1
第貳章、研究方法與材料…………………………………………………8
第一節、細胞株與細胞培養……………………………………………8
第二節、動物實驗設計…………………………………………………9
第三節、動物模型的建立…………………………………………… 10
第四節、動物模型建立之確定……………………………………… 10
一、採血法……………………………………………………… 11
二、抽取骨髓法………………………………………………… 11
第伍節、藥物治療…………………………………………………… 11
第六節、流式細胞儀分析…………………………………………… 12
第七節、組織抹片之觀察…………………………………………… 13
第八節、Trypan blue exclusion 法，計算細胞數……………………14
第九節、細胞週期之分析…………………………………………… 14
第十節、群落(colony)-形成方法…………………………………… 15
第十一節、老鼠體內白血病細胞glutathion含量的觀察……………16
第十二節、利用rhodamine123染劑觀察……………………………16
第參章、實驗結果……………………………………………………… 19
第一節、利用NOD/SCID mice來觀察三氧化二砷對於急性髓性白血病（HL-60）之影響……………………………………………………19
第二節、利用NOD/SCID mice來觀察三氧化二砷對於急性髓性白
血病（primary cell）之影響……………………………………………19
第三節、利用老鼠之間質細胞（MS-5）模擬白血病細胞在骨髓中之細胞週期……………………………………………………………… 20
第四節、利用甲基纖維培養基觀察白血病細胞與間質細胞作用所產生之細胞群落………………………………………………………… 21
第五節、觀察老鼠體內骨髓中之細胞的細胞週期………………… 22
第六節、利用流式細胞儀將小鼠之骨髓細胞分離出來後給予三氧二
砷之治療，並觀察細胞之細胞週期的變化………………………… 22
第七節、觀察小鼠骨髓細胞體內glutathion的含量………………… 23
第八節、利用rodamine123的染劑觀察小鼠是否含有多重抗藥性
（multiple drug resistance）的現象……………………………………24
第肆章、討論與分析…………………………………………………… 26
一、 三氧化二砷對於治療非急性前髓性白血病治療效果之探
討…………………………………………………………………… 26
二、三氧化二砷與小鼠骨髓中微環境之關係…………………… 26
三、小鼠產生抗藥性的原因探討………………………………… 28
參考文獻………………………………………………………………… 32
表格……………………………………………………………………… 37
附圖……………………………………………………………………… 45

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