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研究生:黃秋芬
研究生(外文):C.F. Huang
論文名稱:azatyrosine類似物抗癌活性與機制之研究
論文名稱(外文):A study of antitumor activities and mechanism of azatyrosine anologues.
指導教授:李益謙李益謙引用關係
指導教授(外文):E. I. C. Li.
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
中文關鍵詞:azatyrosine
外文關鍵詞:azatyrosine
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由Streptomyces chibanensis中所分離而得的azatyrosine [D,L-β(5-hydroxypyridin-2yl)-alanine],能夠抑制已活化之人類c-Ha-ras, c- raf, c-ErbB-2致癌基因所引發的轉型NIH3T3細胞(transformed NIH3T3 cells)的生長。然而,azatyrosine的主要缺點是必須在高劑量下方才有活性,推測可能是由於它的胺基酸雙性離子化結構使得通透細胞的能力過低。因此,台大藥學系藥物化學研究者將其結構經過修飾並合成一系列azatyrosine醯胺類衍生物,以增加進入細胞內的能力。本研究試圖探知azatyrosinamides : CHY[D,L-N-propyl 3-(5-Benzyloxypyridin-2-yl)-3-(N-t-butoxycarbonylamido)propionamide]; NPR[D,L-N-cyclohexyl 3-(5-Benzyloxypyridin-2-yl)-3-(N-t-butoxy carbonylamido)propionamide]是否具有毒殺細胞的能力,同時並試圖了解其毒殺細胞的機制為何。我們先選定了NIH3T3 wild type、NIH3T3-ras mutant二種細胞株為研究對象。分別作了azatyrosine, NPR 與CHY對上述兩株細胞之細胞毒性(IC50)測試,軟瓊脂膠生長分析(soft agar growth anylasis ),聚落形成分析(plating efficiency analysis),azatyrosinamides與tyrosine競爭能力分析,DNA合成抑制分析,蛋白質合成抑制分析,細胞核型態觀察(Hoechst stain),Merocyanine 540 染色,細胞週期分析(cell cycle anylasis),及DNA片段分析(DNA fragmentation)等。實驗結果顯示: azatyrosine及NPR與CHY,對NIH3T3 wild type、NIH3T3-ras mutant二種細胞株的細胞毒性(IC50)是有差別的。低濃度的NPR與CHY,除了會抑制NIH3T3-ras mutant細胞在軟瓊脂膠(soft agar)中的生長之外,也可以抑制NIH3T3-wild type細胞形成聚落。 NPR與CHY會抑制DNA與蛋白質的合成,但NPR與CHY不會與tyrosine競爭結合至蛋白質上。根據細胞核型態觀察(Hoechst stain),Merocyanine 540染色,細胞週期分析(cell cycle anylasis),及DNA片段分析(DNA fragmentation)等結果顯示NPR與CHY對於NIH3T3 wild type、NIH3T3-ras mutant二種細胞株所產生的細胞毒殺作用,可能是藉由細胞凋亡(apoptosis)而來的。

Azatyrosine [L-β-(5-hydroxypyrid-2-yl)-alanine], an antibiotic agent isolated from Streptomyces chibanesis, has been reported to exhibit growth inhibitions of c-Ha-ras,c-raf,c-erB-2 transformed NIH3T3 cells. However, to exert its cytotoxicity azatryrosine should be used at a fairly high concentration. We therefore explore the possibility of using its amide analogues as anticancer agents. Two analogues, designed HPW021×314 NPR and HPW021×314CHY, were found to posses low IC50 values against both NIH3T3 wild type and ras-transformed cell lines and, therefore, were subjected to a series of characterizations. The results are as the following. (1) The IC50 values of the two analogues on both cell lines are much lower than that of azatyrosine, being 29- fold to 101-fold lowers on the wild-type and 86- to 226- fold lower on the ras- transformed cells, respectively. (2) The analogues at very low concentrations are enough to inhibit the growth of the ras-transformed cells on soft agar and the colony formation of the wild-type cells on culture dish. (3) The analogues can inhibit both the syntheses of DNA and protein and tyrosine at high concentrations can not reverse the cytotoxcicities of the analogues on both cell lines, suggesting that the analogues do not compete with tyrosine for incorporation into protein. Hence the analogues have a different action mechanism from that of azatyrosine. (5) The results of Hoechst staining, Merocyanine 540 staining, cell cycle analysis, and DNA fragmentation indicate that the analogues may owe their cytotoxicities to their induction of apoptosis on both cell lines.

一. 授權書
二. 考試合格證明
三. 誌謝感言
四. 摘要
A. 中文摘要
B. 英文摘要 i
ii
iv
五. 目錄
六. 圖表錄 v
vii
七. 縮寫表 ix
八. 研究背景 1
九. 研究目的 10
十. 研究方法
A. 細胞培養 11
B. 生長速率分析 11
C. 細胞洋菜膠生長分析 12
D. 細胞毒殺試驗 13
E. 聚落形成能力分析 14
F. DNA片段之純化及電泳分析 15
G. 細胞週期之分析 17
H. 細胞核型態之觀察 18
I. 蛋白質電泳及西方點墨 19
J. 3H-thymidine Incorporation 24
K. 35S-methionine Incorporation 25
十一. 結果 27
十二. 討論 32
十三. 參考文獻 35
十四. 圖表 42
十五. 自述 70

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