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系統識別號 U0026-0812200910442880
論文名稱(中文) 人類血纖維蛋白溶酶原Kringle 1-5區域突變株於血管新生特性之研究
論文名稱(英文) Characterization of the Mutant Kringle 1-5 Domain of Human Plasminogen on Angiogenesis
校院名稱 成功大學
系所名稱(中) 生物化學研究所
系所名稱(英) Department of Biochemistry
學年度 91
學期 2
出版年 92
研究生(中文) 林忠義
研究生(英文) Chung-I Lin
學號 s1690106
學位類別 碩士
語文別 中文
論文頁數 98頁
口試委員 口試委員-張文粲
口試委員-林淑華
指導教授-施桂月
口試委員-吳華林
中文關鍵字 糖基化  血管新生  血管靜止蛋白  離胺酸結合能力 
英文關鍵字 glycosylation  angiostatin  angiogenesis  lysine-binding 
學科別分類
中文摘要 血管新生作用是一個複雜的過程,包括內皮細胞增生、移動、分化和分解細胞外基質,形成管腔,最後長成新的微血管。幾個近來的研究報告指出,腫瘤的生長和轉移依賴血管新生作用,過盛的血管新生屬於癌症病理的一部份,因此防止血管新生的進行是可能的治療策略。

血管靜止蛋白為血纖維蛋白溶脢原的片段,包含其N端3~4個kringle區域,是腫瘤引起血管新生的有效抑制劑。研究報告指出,血管靜止蛋白可增加內皮細胞凋亡,增加focal adhesion kinase活性,以及阻斷內皮細胞移動;它可結合至細胞表面的ATP合成脢,藉由這樣的作用可媒介抗血管新生的效用,和內皮細胞增生、移動的向下調節。然而,kringle結構抗血管新生作用的分子機轉則尚未建立。

為了研究kringle 1-5區域在抗血管新生作用所扮演的角色,我們構築K1-5突變株,包括K1-5 N289A、K1-5 T346A、K1-5 L532R、K1-5 N289A/T346A、K1-5 N289A/L532R、K1-5 T346A/L532R及K1-5 N289A/T346A/L532R,這些突變點關聯糖基化修飾(N289和T346)和lysine結合能力(L532),所有的突變株蛋白以Pichia pastoris表現系統來表現,再經由DEAE Sepharose® FastFlow以及lysine Sepharose® 4B兩支管柱進行純化。在細胞實驗方面,各種K1-5突變株蛋白抑制以bFGF刺激的牛動脈內皮細胞增殖,具有劑量依賴的現象,而K1-5 N289A/T346A/L532R顯示高於天然或重組K1-5的效力,同樣地,在影響內皮細胞的細胞週期,促使細胞凋亡,K1-5 N289A/T346A/L532R亦具較高的效力。在動物實驗方面,包含bFGF和K1-5突變株蛋白的基底膜基質植入小鼠後,有較少的內皮細胞數量和血管結構新生,全身性投予K1-5突變株蛋白有效地阻斷所植入Lewis lung carcinoma的生長,但只有基底膜基質中血紅素含量測量顯示K1-5 N289A/T346A/L532R突變株蛋白有最高的效力。總之,重組K1-5蛋白的抗血管新生能力可藉由減少糖基化修飾和增強K5的lysine結合能力而增加。
英文摘要 Angiogenesis is a complex multistep process that includes endothelial cell proliferation, migration, and differentiation, degradation of extracellular matrices, tube formation, and sprouting of new capillary branches. Several recent studies show that tumor growth and metastasis are angiogenesis-dependent. Excessive angiogenesis is a part of the pathology of cancer, and preventing this process is a promising therapeutic strategy.

Angiostatin, a plasminogen fragment containing 3-4 N-terminal kringle domains, is a potent inhibitor of tumor-induced angiogenesis. Angiostatin has been reported to increase endothelial cell apoptosis, increase the activity of focal adhesion kinase, and block the migration of endothelial cells. It has been reported that angiostatin binds to ATP synthase on the cell surface and that binding of angiostatin to ATP synthase may mediate the anti-angiogenic effects of angiostatin and the down-regulation of endothelial cell proliferation and migration. However, the molecular mechanism underlying the anti-angiogenic actions of the kringle structures has not been established.

To investigate the role of kringle 1-5 domain in anti-angiogenesis, K15 mutants were made, including K1-5 N289A, K1-5 T346A, K1-5 L532R, K1-5 N289A/T346A, K1-5 N289A/L532R, K1-5 T346A/L532R, and K1-5 N289A/T346A/L532R. These residues located in kringle 1-5 domain involved mainly in glycosylation (residues N289 and T346) and lysine binding (residue L532). All the mutant proteins were expressed by Pichia pastoris expression system and then purified by DEAE Sepharose® and lysine Sepharose® columns. In vitro assay, various K1-5 mutant proteins inhibited the proliferation of bovine arterial endothelial (BAE) cells stimulated by bFGF in a dose-dependent manner and K1-5 N289A/T346A/L532R exhibited a higher potency than that of native or recombinant K1-5. Also, they inhibited cell cycle progression of BAE cells and induced apoptosis, and the K1-5 N289A/T346A/L532R was more effective than native or recombinant K1-5. In animal models, the mutant K1-5-treated Matrigel® plugs, which had been stimulated with bFGF, had reduced numbers of endothelial cells and vessel structures. Systemic administration of the mutant K1-5 potently blocked the growth of inoculated Lewis lung carcinoma. Hemoglobin assay of Matrigel® plugs showed K1-5 N289A/T346A/L532R had the highest potency. Taken together, anti-angiogenesis ability of recombinant kringle 1-5 could be further enhanced by altering glycosylation sites and increase lysine-binding affinity of kringle 5.
論文目次 中文摘要 ...... 1
英文摘要 ...... 3
目錄 ...... 5
表、圖、附錄目錄 ...... 7
縮寫檢索表 ...... 9
緒論 ...... 11
藥品 ...... 17
儀器 ...... 21
材料及方法 ...... 23
一、K1-5雙點或三點突變株之構築 ...... 23
 1.限制酶處理 ...... 23
 2.去除載體DNA上5'端之磷酸根 ...... 23
 3.瓊脂膠電泳 ...... 24
 4.以電泳法回收DNA ...... 25
 5.接合反應 ...... 26
 6.大腸桿菌E. coli之形質轉移 ...... 27
 7.小量質體DNA抽取 ...... 29
 8.核酸定序分析 ...... 31
 9.長期菌種保存 ...... 31
二、重組蛋白的表現與純化 ...... 31
 1.酵母菌Pichia pastoris之形質轉移 ...... 31
 2.小量表現 ...... 34
 3.點狀印漬 ...... 35
 4.長期菌種保存 ...... 37
 5.大量表現 ...... 37
 6.重組蛋白質之純化 ...... 40
 7.蛋白質電泳 ...... 41
 8.西方點墨法 ...... 43
三、重組蛋白之功能分析 ...... 44
 1.Bovine aortic endothelial cells (BAE cells)之培養 ...... 44
 2.Lewis lung carcinoma cells (LLC cells)之培養 ...... 45
 3.細胞計數 ...... 46
 4.抑制內皮細胞增殖之功能分析 ...... 47
 5.細胞週期分析 ...... 48
 6.小鼠皮下植入基底膜基質之分析 ...... 49
 7.小鼠體內抗腫瘤活性之功能分析 ...... 51
結果 ...... 53
討論 ...... 57
參考文獻 ...... 61
表 ...... 68
圖 ...... 69
附錄 ...... 93
自述 ...... 98
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楊晉姿 (2001) 重組人類血纖維蛋白溶脢原K1-5及其突變株之功能分析,國立成功大學生物化學研究所碩士論文
吳柏義 (2002) 人類血纖維蛋白溶脢原之kringle結構區對血管新生抑制作用之研究,國立成功大學生物化學研究所碩士論文
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