臺灣博碩士論文加值系統

端粒為真核細胞染色體末端的核酸蛋白結構，正常的端粒結構具有維護染色體完整性等功能。在正常的體細胞內，由於細胞的末端複製問題，在每一次細胞分裂後端粒會縮短約50-60 bp，當端粒縮短至關鍵長度，細胞將進入衰老並導致凋亡。而端粒酶是由特定的反轉錄酵素與RNA模板組成，具有合成端粒序列及維持端粒長度的功能，端粒酶在近年常做為人類癌症的指標，由於正常的體細胞不具有端粒酶活性，但大多數的人類腫瘤細胞則具有端粒酶活性及較正常細胞短的端粒，對於腫瘤細胞與正常細胞間有選擇性，因此以抑制端粒酶為標靶來治療癌症是相當具有吸引力的研究。許多研究指出，具平面芳香環的結構化合物對於抑制端粒酶有很好的效果，例如以蒽菎為主體的衍生物，能夠嵌入端粒末端的DNA序列，形成穩定的四股複合體結構 (G-quadruplex structures)，使端粒酶無法延長端粒，抑制端粒酶活性進而達到抗癌的效果。本論文合成一系列的anthra[1,2-d]imidazole-6,11-dione衍生物，來探討此新穎化合物對端粒酶活性的抑制。在藥理活性試驗方面主要是端粒序列複製法 (TRAP assay) 評估化合物對端粒自行形成的特殊結構G-quadruplex的穩定情形、MTT assay評估細胞存活率及利用SEAP assay偵測PhTERT的表現。本研究希望從此系列化合物中，尋找具有端粒酶抑制作用的化合物，期望能繼續發展成為有潛力的抗癌藥物。

Telomere is the nucleoprotein structure at the end of eukaryotic chromosomes, and functional telomeres are essential for continued cell proliferation. Because of the end replication problem, telomere is shortened by 50-60 bases whenever cell division happens, and this erosion induces apoptosis when its length became critically short. The enzyme telomerase is able to perform this function of length extension, since it is a specialized reverse transcriptase with an endogenous RNA template on which successive telomeric repeats are synthesized. It has been approximately a decade since telomerase was described as an almost universal marker for human cancer. Most human tumors not only express telomerase but also have very short telomeres, whereas telomerase activity is either reduced or absent in normal tissues, making the inhibition of telomerase an attractive target for cancer therapeutics. In the recent research shown that inhibition of telomerase can be achieved with appropriately planar aromatic structure such as anthraquinone derivatives. This inhibition is believed to occur as a result of the stabilization of telomeric DNA by these compounds as folded G-quadruplex structures, which are known to inhibit telomerase activity. A series of anthra[1,2-d]imidazole-6,11-dione derivatives were synthesized for the development of human telomerase inhibitors as potential anticancer agents, which will be evaluated for their effects by TRAP assay, cell proliferations and cytotoxicity by MTT assay, hTERT expression by SEAP assay.

第一章、緖論 -------------------------------------------------------------------------------- 1
第一節、前言 -------------------------------------------------------------------------- 1
壹、端粒 -------------------------------------------------------------------------- 2
貳、端粒酶 ----------------------------------------------------------------------- 6
參、端粒酶與癌症 -------------------------------------------------------------- 8
肆、以端粒為標靶的端粒酶抑制劑 --------------------------------------- 10
伍、Anthraquinone衍生物簡介 --------------------------------------------- 17
陸、Anthraquinone衍生物與topoisomerase II的抑制 ------------------- 19
第二節、Anthraquinone衍生物文獻回顧 ---------------------------------------- 22
第三節、藥物設計原理 ------------------------------------------------------------- 30
第二章、材料與方法 ---------------------------------------------------------------------- 34
第一節、化學合成 ------------------------------------------------------------------- 34
壹、實驗儀器 ------------------------------------------------------------------ 34
貳、試藥來源 ------------------------------------------------------------------ 34
參、化學合成實驗方法 ------------------------------------------------------ 36
第二節、藥理活性實驗 ------------------------------------------------------------- 39
壹、端粒序列複製法 --------------------------------------------------------- 39
貳、MTT比色法抗癌活性測試 --------------------------------------------- 43
参、分泌型鹼性磷酸酶分析法 --------------------------------------------- 46
肆、美國癌症學會癌細胞株毒殺試驗 ------------------------------------ 50
第三章、實驗結果 ------------------------------------------------------------------------- 52
第一節、化學合成結果 ------------------------------------------------------------- 52
第二節、藥理實驗結果 ------------------------------------------------------------- 93
壹、TRAP assay篩選結果 --------------------------------------------------- 93
貳、MTT assay與SEAP assay篩選結果 ----------------------------------- 96
参、美國癌症學會癌細胞株毒殺試驗結果 ---------------------------- 102
第四章、討論與結論 -------------------------------------------------------------------- 105
第一節、化學合成討論 ----------------------------------------------------------- 105
壹、1,2-Diaminoanthraquinone的環化反應 ----------------------------- 105
貳、[1,2-d]咪唑蒽醌衍生物討論 ----------------------------------------- 108
参、[1,2-d]咪唑蒽醌衍生物NMR圖譜討論 ---------------------------- 113
第二節、藥理實驗結論 ----------------------------------------------------------- 114
壹、TRAP assay結論 -------------------------------------------------------- 114
貳、MTT assay與SEAP assay結論 --------------------------------------- 115
第五章、參考文獻 ----------------------------------------------------------------------- 122

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