臺灣博碩士論文加值系統

端粒酶在細胞的永生性及抗癌過程都扮演重要的角色，在腫瘤細胞中端粒酶活性常會被活化。本實驗室長久以來尋找並探討端粒酶抑制劑作為抗癌藥物的可能性，近日發現一極具有潛力的藥劑 N-(1-Pyrenyl)maleimide（NPM）對人類急性 T 細胞淋巴性白血病細胞（T-ALL）Jurkat 細胞具有選擇性毒殺作用的特性。本研究建立活體螢光白血病小鼠模式，探討 NPM 在活體內對白血病之抗癌活性。首先我們建立穩定表達螢光素酶 Jurkat-Luc T-ALL 細胞以及可穩定表達近紅外光 Jurkat-IR T-ALL 細胞，也證實 Jurkat、Jurkat-Luc 及 Jurkat-IR 細胞對於 NPM 的耐受性有相似的趨勢。將 Jurkat-Luc 以皮下注射建立動物模式後給予 4 mg/kg 的 NPM，由非侵入式活體分子影像系統（IVIS）、H&E 染色以及免疫組織化學染色法等結果顯示：NPM 可抑制 Jurkat 細胞在裸鼠體內的生長。將臨床抗白血病藥物環磷醯胺結合 NPM 在 T-ALL 細胞及動物實驗試驗中皆有協同作用。綜合上述實驗結果，我們證實 NPM 在裸鼠活體內有抗白血病的活性。

Telomerase activity is normally not detectable in most somatic cells, but is activated in the vast majority of cancer cells. Therefore, inhibition of telomerase has been viewed as a promising anticancer approach due to its specificity for cancer cells. N-(1-pyrenyl) maleimide (NPM) has been identified as a telomerase inhibitor in a cell-free system by our group. We also demonstrated that N-(1-pyrenyl) maleimide induces apoptosis in Jurkat cells (T-cell acute lymphoblastic leukemia, T-ALL) and displays the greatest differential cytotoxicity against hematopoietic cancer cells. This study was to evaluate the effects of apoptosis by NPM on the development of T-ALL using a bioluminescent mouse model. Jurkat cells were transfected with luciferase-expressing or near-infrared-expressing plasmid to establish bioluminescent T-ALL cells (Jurkat-Luc and Jurkat-IR). The luciferase–expressing T-ALL cells were mixed with matrigel and injected subcutaneously into the nude mice. Bioluminescent imaging showed a significant reduction in tumor burden in mice from NPM treatment group. Combined treatment with NPM and cyclophosphamide (CTX), a leukemia classical drug, enhanced to inhibit cell viability and the growth of xenograft tumors derived from Jurkat cells. Our results indicated that NPM is effective treatment for T-ALL.

長庚科技大學學位論文授權書
碩士學位論文指導教授推薦書
碩士學位論文口試審定
中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 vii
第一章　緒論 1
第一節　癌症（Cancer） 1
第二節　白血病（Leukemia） 1
第三節　白血病目前治療 3
第四節　急性 T 細胞淋巴性白血病（T-cell acute lymphoblastic leukemia; T-ALL）生物活體螢光動物模式( Bioluminescent mouse model ) 5
第五節　N-(1-Pyrenyl) maleimide 目前的應用 6
第六節　N-(1-Pyrenyl) maleimide 應用於白血病的研究 6
第二章　材料與方法 8
第一節　細胞培養 8
第二節　質體（Plasmids） 8
第三節　質體轉形（Transformation） 8
第四節　確認質體 9
第五節　質體放大 9
第六節　電穿孔法（Electroporation） 9
第七節　選殖細胞（Selection of stable cell lines） 10
第八節　螢光素酶試驗（Luciferase assay） 10
第九節　Jurkat-IR 細胞近紅外光（Near-infrared; NIR）表現 11
第十節　試劑 11
第十一節　人類周邊血液淋巴球的製備與培養 11
第十二節　細胞存活率試驗－MTS assay 12
第十三節　細胞存活率試驗－Trypan blue assay 12
第十四節　活體螢光（Bioluminescent）白血病動物模式：皮下（subcutaneous xenografts）模式 13
第十五節　動物模式體內螢光素酶（Luciferase）監測 13
第十六節　動物模式體內近紅外光（Near-infrared; NIR）監測 13
第十七節　傳統抗白血病藥物合併 NPM 療效 14
第十八節　動物藥物給予（Treated drug in vivo） 14
第十九節　H&E 染色 14
第二十節　免疫組織化學染色法（Immunohistochemistry; IHC） 15
第二十一節　藥物聯合作用指數（Drug combination index; CI） 15
第二十二節　統計分析（Statistical analysis） 16
第三章　結果 17
第一節　確認質體 17
第二節　建立活體影像系統細胞株 17
第三節　確認 Jurkat-Luc 細胞中螢光素酶的表現量 17
第四節　確認 Jurkat-IR 細胞近紅外光的表現量 18
第五節　NPM 對於 Jurkat 細胞、Jurkat-Luc 及 Jurkat-IR 細胞毒殺作用 18
第六節　建立近紅外光（Near-infrared）表現活體影像系統動物模式 19
第七節　建立螢光素酶（Luciferase）表現活體影像系統動物模式以探討NPM 抑制白血病細胞之活體內腫瘤生成能力 20
第八節　NPM 合併臨床白血病用藥於白血病細胞之協同作用（Synergism effect） 22
第九節　NPM 合併臨床血癌用藥抑制白血病細胞之活體內腫瘤生成能力 23
第四章　討論 25
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