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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張智芬(Zee-Fen Chang) | |
dc.contributor.author | Chuan-Mei Hu | en |
dc.contributor.author | 胡春美 | zh_TW |
dc.date.accessioned | 2021-06-13T16:30:05Z | - |
dc.date.available | 2005-08-03 | |
dc.date.copyright | 2005-08-03 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38309 | - |
dc.description.abstract | 在dTTP生成的過程中,thymidylate kinase (TMPK) 扮演重要的角色,它催化來自新合成以及回收利用路徑的dTMP,使之轉變成dTDP,進而形成dTTP,因此TMPK對於dTTP的生成相當重要,但目前對於它在細胞週期中的調控機轉,所知甚少。
為了能夠深入探究TMPK在細胞中的調控機制,我的研究第一步就是製備能夠辨識人類TMPK (hTMPK) 的多株抗體。結果顯示,我所製備的anti-hTMPK多株抗體可以專一性地辨識多種人類細胞內生性hTMPK蛋白質,另外,進一步利用純化的anti-hTMPK抗體,執行免疫染色分析,可偵測hTMPK蛋白質在正常纖維母細胞中分佈的位置。 除此之外,為了了解調控hTMPK蛋白質降解的機制,我們運用定點突變的技術,突變hTMPK蛋白質胺基酸序列中,可能和蛋白質降解有關的胺基酸,產生許多hTMPK的異變體,進一步分析這些異變體的性質,發現其中特一異變體 (36RAEL39 →36AAEA39) 具有較高的TMPK催化活性,而變異催化位置的胺基酸 (D15 →R15、R97 →A97、R143 →A143、 F146 →A146) 或是與蛋白質降解作用相關訊號的胺基酸變異 (5RGAL8 →5AGAA8、109KEN111 →109AAA111 ),則降低 hTMPK蛋白質的酵素活性。 在本研究中,我也利用RNAi的技術抑制hTMPK的表現量,探討是否可藉此影響dTTP pool,而抑制癌細胞生長,並提高基因毒性藥物之毒殺效益。實驗結果顯示,無論是利用合成的siRNA或shRNA都能抑制HCT116結腸癌細胞中hTMPK的表現量,並降低細胞中dTTP的含量,進而抑制HCT116結腸癌細胞的生長速率,而且同時增加DNA損壞藥物 - Doxorubicin對細胞的毒殺效益。這研究對未來抗癌治療提供了理性設計的基礎。 | zh_TW |
dc.description.abstract | Human thymidylate kinase (hTMPK) catalyses a critical step in the biosynthesis of deoxythymidine tirphosphate (dTTP) from either de novo or salvage pathway. However, information regarding the regulatory mechanism controlling hTMPK expression in the cell cycle has been lacking.
In this study, I generated polyclonal antibody specifically against hTMPK, and confirmed it's specificity by antigen neutralization experiment. This antibody can specifically recognize endogenous hTMPK in extracts from various human cell lines. Using a purified antibody, we were able to detect the subcellular localization of hTMPK by immunostaing. We generated various mutations in the putative degradation signal motifs and catalytic domains of hTMPK. The purified recombinant proteins of hTMPK variants were subjected to enzymatic assay and kinetic analysis. We found that some mutations of the residues within the catalytic site (D15 →R15)and destruction motifs (5RGAL8 →5AGAA8, 36RAEL39 →36AAEA39, and 109KEN111 →109AAA111) caused change of kinetic properties. In this study, I also knockdown hTMPK expresion by small interference RNA to test whether decrease of cellular dTTP can sensitize cancer cells to DNA damage-induced cell death, thereby promoting the efficacy of genotoxic agents in cancer cell line. My results showed that prevention of hTMPK expression by synthetic siRNA or shRNA decreased the dTTP pool and suppressed the growth rate of HCT116 colon cancer cells. This also sensitized cells to Doxorubicin-induced apoptosis in HCT116 cells. Through these experiments, my results thus provide a rational basis for the design of therapeutic strategies for cancer. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T16:30:05Z (GMT). No. of bitstreams: 1 ntu-94-R92442001-1.pdf: 2544067 bytes, checksum: 5757cc74003706cfb490c9c00d6d9f76 (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………2 前言………………………………………………………………………3 緒論………………………………………………………………………4 實驗材料和方法…………………………………………………………8 實驗結果 ………………………………………………………………24 第一部分: hTMPK多株抗體的製備 第二部分: hTMPK variants二級結構和酵素活性的分析 第三部分: 藉由siRNA的技術抑制hTMPK蛋白的表現,觀察hTMPK對於細胞生長、dTTP pool size的調控和 DNA受損訊息等反應的關係。 討論 ……………………………………………………………………32 圖表 ……………………………………………………………………36 參考文獻 ………………………………………………………………66 附錄 ……………………………………………………………………76 | |
dc.language.iso | zh-TW | |
dc.title | 人類胸腺嘧啶核酸激酶功能研究 | zh_TW |
dc.title | Functional analysis of human thymidylate kinase | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李芳仁,李財坤,游偉絢 | |
dc.subject.keyword | 胸腺嘧啶核酸激酶, | zh_TW |
dc.subject.keyword | thymidylate kinase, | en |
dc.relation.page | 79 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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