臺灣博碩士論文加值系統

第一型聚腺嘌呤雙磷酸核糖聚合酶 (poly(ADP-ribose) polymerase-1, PARP-1) 是豐富的核內蛋白，是一個與染色質結構及DNA修復有關的多功能調控蛋白。目前已經證實有許多癌症都會高度表現第一型聚腺嘌呤雙磷酸核糖聚合酶，所以聚腺嘌呤雙磷酸核糖聚合酶抑制劑應是有效治療腫瘤的藥物。生殖腺BRCA基因突變的情況下，若媒介第一型聚腺嘌呤雙磷酸核糖聚合酶活化修復受損癌細胞DNA，會使乳癌細胞對聚腺嘌呤雙磷酸核糖聚合酶抑制劑敏感性增加。BRCA基因突變也在非小細胞肺癌中發現。我們找尋並發展帶有蒽醌 (anthraquinone) 的藥物核心且部分媒介聚腺嘌呤雙磷酸核糖聚合酶而對非小細胞肺癌有細胞毒性的領導化合物。另一方面，目前已由基因層面證實在高效率重新編程的細胞 (efficient reprogramming clones) 中，與修復DNA /改變組蛋白構型的基因表現量上升。我們發現老鼠胚胎纖維母細胞 (mouse embryonic fibroblast, MEFs) 與誘導性多能幹細胞 (induced pluripotent stem cells, iPS cells) 第一型聚腺嘌呤雙磷酸核糖聚合酶表現量不同。在重新編程的過程中，處理聚腺嘌呤雙磷酸核糖聚合酶抑制劑及我們證實具有抑制聚腺嘌呤雙磷酸核糖聚合酶的蒽醌衍生物 (NSC747854) ，可能會抑制細胞群落形成的效率並降低鹼性磷酸酶 (alkaline phosphatase) 的分泌。

The abundant nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1), a multifunctional regulator of chromatin structure and DNA repair. High expression of PARP-1 has been indicated in several cancer types, so inhibitors of PARP-1 might be effective as therapeutic agents for the treatment of tumors. Germline mutations in the genes BRCA sensitize breast cancer cells to PARP inhibitors in a PARP-1-dependent manner. BRCA mutation also has been observed in non-small-cell lung cancers (NSCLCs). We screen and develop some lead compounds with anthraquinone-based pharmacophore, which have cytotoxicity to NSCLCs partly via inhibiting PARP activity. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient reprogramming clones. We found expression of PARP-1 is different between mouse embryonic fibroblast (MEFs) and induced pluripotent stem (iPS) cells. During reprogramming process, treating with PARP inhibitor and our found one putative PARP inhibitor of anthraquinone derivatives (NSC747854) might suppress the efficiency of colony formation and decrease secretion of alkaline phosphatase.

總目錄
頁
正文目錄……………………………………………………………………… II
表目錄………………………………………………………………………… IV
圖目錄………………………………………………………………………… V
中文摘要…………………………………………………………………………V II
英文摘要…………………………………………………………………………V III

正文目錄
頁
第一章 緒論…………………………………………………………………1
第一節、前言..………………………………………………………………1
壹、 聚腺嘌呤雙磷酸核糖聚合酶 (poly(ADP-ribose) polymerase, PARP)…………………………………………………………………………………1
貳、 PARP抑制劑 (PARP inhibitors)…………………………………………………3
參、 非小細胞肺癌 (non-small cell lung cancer, NSCLC)…6
肆、 重新編程 (Reprogramming)………………………………………………………………7
第二節、研究動機與目的……………………………………………………12
第二章 研究材料及方法……………………………………………………14
壹、 細胞培養 (Cell culture)………………………………………………………………14
貳、 細胞計數 (Cell counting)……………………………………………………………14
參、 細胞存活率分析 (MTT assay)…………………………………………………………15
肆、 聚腺嘌呤雙磷酸核糖聚合酶活性抑制測試 (PARP inhibitory assay)…………………………………………………………………………………15
伍、 質體製備 (Plasmid preparation)………………………………………………16
陸、 西方墨點法 (Western blotting)…………………………………………………18
柒、 即時定量聚合酶鏈鎖反應 (quantitative polymerase chain reaction, qPCR)…………………………………………………………………………………20
捌、 小鼠胚胎纖維母細胞之建立 (Establish mouse embryonic fibroblast)…………………………………………………………………………………21
玖、 餵養細胞之製備 (Preparation of feeder cells)……………22
壹拾、 重新編程 (Reprogramming)………………………………………………………………23
壹拾壹、 PARP抑制劑處理 (PARP inhibitor treatment)……………………24
壹拾參、 鹼性磷酸酶染色 (Alkaline phosphatase staining, AP staining)……………………………………………………………………………………24
壹拾肆、 化學合成……………………………………………………………24
第三章 研究結果………………………………………………………………29
壹、 挑選化合物進行PARP抑制劑篩選…………………………………29
貳、 化合物於細胞實驗結果……………………………………………34
參、 PARP活性抑制測試…………………………………………………40
肆、 PARP抑制劑於重新編程過程處理之影響…………………………43
伍、 sh PARP-1於重編程過程處理之影響……………………………53
第四章 實驗討論………………………………………………………………56
第五章 參考文獻………………………………………………………………61

表目錄
頁
表1、美國國家癌症研究中心 (National cancer institute, NCI) 於非小細胞肺癌 (A549、H322M、HOP-92、H460及H23) 進行SRB assay的各種濃度結果…………………………………………………………………………………33

圖目錄
頁
圖1、由交互作用 (interaction) 層面挑選可能為PARP抑制劑之化合物…31
圖2、兩種癌細胞處理候選化合物及PARP抑制劑後之存活率…………………36
圖3、候選化合物在癌細胞與正常細胞之IC50比較……………………………38
圖4、PARP活性抑制試驗結果……………………………………………………41
圖5、處理不同濃度PARP抑制劑 (3-AB) 於重新編程後第六天MEF細胞型態……………………………………………………………………………………45
圖6、處理不同濃度PARP抑制劑 (PJ34) 於重新編程後第六天MEF細胞型
態……………………………………………………………………………46
圖7、處理不同濃度PARP抑制劑 (3-AB) 於重新編程後第十七天MEF細胞型態……………………………………………………………………………………47
圖8、處理不同濃度PARP抑制劑 (PJ34) 於重新編程後第十七天MEF細胞型態……………………………………………………………………………………48
圖9、處理不同濃度PARP抑制劑 (3-AB) 鹼性磷酸酶活性試驗………………49
圖10、處理不同濃度PARP抑制劑 (PJ34) 鹼性磷酸酶活性試驗……………50
圖11、處理不同濃度PARP抑制劑候選化合物 (NSC747854) 鹼性磷酸酶活
性試驗………………………………………………………………………………51
圖12、確認shPARP-1調降 (knockdown) 效率………………………………54
圖13、送入shPARP-1重新編程之鹼性磷酸酶活性試驗………………………55

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