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研究生:杜修齊
研究生(外文):Tu Hsiu Chi
論文名稱:二種血管形成抑制因子的表現及特性分析
論文名稱(外文):Expression and Characterization of Two Angiogenesis Inhibitors
指導教授:蕭世裕
指導教授(外文):Shah-Yu Shaw
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:57
中文關鍵詞:血管形成血管形成
外文關鍵詞:AngiogenesisAngiostatin
相關次數:
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血管形成 (Angiogenesis),對原發性腫瘤的生長及轉移極為關鍵,因此,開啟了惡性腫瘤的新治療法。
血管形成(Angiostatin,K1-4)是一種有效的血管形成抑制因子,由human plasminogen的前四個Kringle組成 (K1-4),能抑制腫瘤的生長。已知K1-4能抑制微血管內皮細胞的增生及遷移,但是單獨的Kringle 4抑制內皮細胞增生的能力則相當微弱,那麼,是否K1-4為Plasminogen具抑制腫瘤活性的最小片段?
本論文的目的在同時表現兩種嵌合蛋白,一種包含了人類Plasminogen的Kringle 1至Kringle 4及人類IgG的CH2至CH3片段(K1-4/IgG)、另一種包含了Kringle 1至Kringle 3及IgG的CH2至CH3片段 (K1-3/IgG)。冀望藉由嵌合蛋白的設計,獲得二聚體形式的蛋白質分子,以提高其抑制微血管內皮細胞增生或遷移的能力,同時,IgG部分有可能延長其在血液中的半生期並且有助於蛋白質的快速純化。
本實驗在CHO cells中表現K1-4/IgG及K1-3/IgG二種嵌合蛋白,確保糖基化的蛋白質與自然存在於人體內之蛋白質的本質是最相近的,CHO cells生產的嵌合蛋白可分泌至細胞培養液中,而簡化純化步驟。純化後之嵌合蛋白以蛋白質電泳分析,得K1-4/IgG在非還原態下,分子量大約為170 KD,在還原態下,分子量為100 KD;而K1-3/IgG在非還原態下,分子量約為150 KD,在還原態下,分子量為80 KD,表示K1-4/IgG及K1-3/IgG二種嵌合蛋白的確為二聚體的蛋白質分子。實驗也證明嵌合蛋白的純化可用一種純化方法來完成。
在抑制內皮細胞增生的測試中,得K1-4/IgG的IC50=1200 nM,而K1-4及K1-3/IgG在濃度1500 nM時,對內皮細胞的增生完全沒有抑制效果;在抑制內皮細胞遷移的測試中,得K1-4/IgG的IC50=500 nM、K1-4的IC50=1000 nM及K1-3/IgG的IC50<500 nM。顯示二聚體K1-4/IgG比單體K1-4具備更佳的抑制活性。
本文嘗試著以內皮細胞的Cell line (EJG及BAE)來作為活體外測試的細胞來源,故EJG及BAE對嵌合蛋白的敏感度可能不如前人所採用的Primary cell。

Angiogenesis is crucial for the growth and metastasis of primary tumors. Consequently, the angiogenic process triggers the development of therapies for the treatment of malignant disease.
Angiostatin is one of angiogenic inhibitors, consists of the first four kringle domains(K1-4)of human plasminogen and inhibits the growth of tumors. It was known that K1-4 inhibited the proliferation and migration of bovine capillary endothelial cells, but kringle 4 alone had only marginal anti-proliferative activity. Was K1-4 the minimal fragment of plasminogen that maintained anti-tumor activity?
We expressed two chimeric proteins that consisted of full-length human angiostatin (K1-4) or a truncated form of angiostatin (K1-3) fused with IgG. The chimeric protein (K1-4/IgG) contained kringle1-4 of human plasminogen and CH2-CH3 of human IgG. While the other (K1-3/IgG) contained kringle 1-3 of human plasminogen and CH2-CH3 of human IgG. By designing these chimeric proteins, we hoped to obtain a dimer of K1-4 or a dimer of K1-3 which might enhance its ability in anti-proliferation or anti-migration of endothelial cells. The IgG portion of the chimeric proteins might prolong its half-life in circulation and might facilitate the purification process of proteins.
We planed to express K1-4/IgG and K1-3/IgG proteins in CHO cells to make sure the glycosylated pattern were similar to that of native human proteins, and the secretion of both proteins to the cell culture medium might simplify the purification steps. The purified chimeric proteins were analyzed by SDS-PAGE. K1-4/IgG had a molecular weight about 170 KD under non-reducing condition and 100 KD under reducing condition. K1-3/IgG had a molecular weight about 150 KD under non-reducing condition and 80 KD under reducing condition. The results showed that these two chimeric proteins indeed formed dimer and could be purified by one step.
In anti-proliferation assay, it was shown that K1-4/IgG inhibited the proliferation of bovine capillary endothelial cells with a IC50 of 1200 nM, but both K1-4 and K1-3/IgG had no such inhibition activity. In anti-migration assay, K1-4/IgG inhibited the migration of bovine endothelial cells with a IC50 of 500 nM, but K1-4 was 1000 nM. So K1-4/IgG dimer had better inhibition activity than K1-4 monomer.
In this report, we used bovine capillary endothelial cell line (EJG及BAE) instead of primary endothelial cells, so the cells might have less sensitivity in response to these chimeric proteins.

中文摘要 i
英文摘要 iii
誌謝 v
目錄 vi
表目錄及圖目錄 vii
緒論 1
目的 9
材料與方法 10
結果與討論 20
結論 29
參考文獻 30
表 目 錄 及 圖 目 錄
(表1) 目前已知的血管形成調控蛋白 33
(表2) K1-4的N-端定序結果 33
(圖1) 血管形成刺激因子、抑制因子間平衡狀態與
血管形成之關聯 34
(圖2) Angiostatin 的組成及結構 34
(圖3) 二聚體K1-4/IgG嵌合蛋白的組成 35
(圖4) K1-4/IgG表現載體的設計 36
(圖5) K1-3/IgG表現載體的設計 37
(圖6) 不同Clone間,K1-4/IgG嵌合蛋白之表現量 38
(圖7) 不同Clone間,K1-3/IgG嵌合蛋白之表現量 39
(圖8) 可生產K1-4/IgG之細胞株間基因增殖的比較 40
(圖9) 可生產K1-3/IgG之細胞株間基因增殖的比較 41
(圖10) 以銀染法進行K1-4的分析 42
(圖11) 以銀染法進行K1-4/IgG嵌合蛋白的分析 43
(圖12) 以銀染法進行K1-3/IgG嵌合蛋白的分析 44
(圖13) MTS tetrazolium及其產物(formazan)的結構 45
(圖14) EJG內皮細胞數目對490nm吸收值的影響 46
(圖15) BAE內皮細胞數目對490nm吸收值的影響 46
(圖16) 嵌合蛋白抑制EJG內皮細胞增生的測試 47
(圖17) 嵌合蛋白抑制BAE內皮細胞增生的測試 47
(圖18) BAE內皮細胞的遷移 48
(圖19) 嵌合蛋白抑制BAE內皮細胞遷移的測試 49

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