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研究生:吳柏義
研究生(外文):Po-Yi Wu
論文名稱: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
論文名稱(外文):Study on the Inhibitory Effect of Kringle Domains of Human Plasminogen on Angiogenesis
指導教授:施桂月
指導教授(外文):Guey-Yueh Shi
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:83
中文關鍵詞:血管新生內皮細胞血纖維蛋白溶脢原蛋白純化腫瘤生長
外文關鍵詞:angiostatinkringle domainplasminogenprotein purificationturmor growth
相關次數:
  • 被引用被引用:6
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血管新生過程是由已存在的血管長出新血管的過程,此過程包括了細胞間質的分解,內皮細胞的增生、遷移及分化,最後長出新的血管。而腫瘤的生長需要血管新生的參與,因此調控血管新生作用的因子可以影響腫瘤的生長。1994年Folkman等人在患有Lewis lung carcinoma的老鼠的血漿及尿液中發現一種可以抑制老鼠腫瘤生長時血管新生的蛋白質存在,並命名為angiostatin;angiostatin由老鼠的血液中純化出來後,經胺基酸序列分析發現其為老鼠血纖維蛋白溶脢原(plasminogen)之內部片段,分子量為38 kda,包含kringle 1-4 domain(K1-4)。Angiostatin可以專一地抑制由bFGF所誘發之內皮細胞增生,也可以在in vivo下抑制腫瘤之生長。1997年Cao等人發現人類血纖維蛋白溶脢原之krigle 5 domain具有比angiostatin更強的效果;之後Cao與本實驗室合作,使用由人類血纖維蛋白溶脢原切割下來的kringle 1-5 domain(K1-5),亦發現K1-5可抑制內皮細胞增生及腫瘤生長,且效果更勝kringle 5及angiostatin。
為探討人類血纖維蛋白溶脢原之kringle結構區在抑制血管新生作用上之效果,我們使用酵母菌表現系統表現K1-5及其相關片段K2-5和K1-3+5,以純化後之重組蛋白進行抑制內皮細胞生長之分析。
實驗室先前已有可以正確表現重組蛋白(K1-5,2-5,K1-3+5)之酵母菌菌株,經水浴槽或發酵槽培養並誘導表現後,各片段之重組蛋白皆順利分泌至培養液中。我們將培養液濃縮,使用DEAE Sepharose CL-6B以及lysine-Sepharose兩支管柱進行純化,再觀察純化後的重組蛋白:K1-5,K2-5以及K1-3+5抑制內皮細胞增殖之情形。結果發現各片段蛋白皆具有抑制內皮細胞BEC的生長的活性,使用人類內皮細胞HMEC-1進行實驗亦可以得到類似的結果;而在相同的濃度處理下,以K1-5的活性較好。另外我們嘗試在低血清濃度下對BEC處理重組蛋白,除觀察到細胞有型態上的變化,亦利用propidium iodide染色及流式細胞儀分析內皮細胞之細胞週期變化,發現K1-5重組蛋白會影響細胞週期分布,並造細胞計畫性死亡。另外實驗室先前已將人類K1-5及其相關片段K1-4,K1-3+5及plasminogen構築到真核表現載體pEGFP-N1,我們將這些片段轉染至Lewis lung carcinoma cells(LLC)內,經過篩選後,利用dot blotting偵測培養液,以確認得到穩定表現分泌K1-5相關片段蛋白的細胞株。首先觀察轉染細胞之生長,發現腫瘤細胞所分泌的K1-5相關片段蛋白並不會抑制腫瘤細胞本身的生長。接著將穩定分泌K1-5之腫瘤細胞以皮下注射植入小鼠體內,比較各細胞株在初期腫瘤生長上差異,在21天的觀察期中,發現可表現K1-5及相關片段之腫瘤細胞較控制組(LLC)有較慢的腫瘤生長速度及較小的腫瘤形成,在兩次的實驗中,觀察到各片段蛋白抑制能力約為27 %至62 %,而K1-5有較佳的抑制效果。
Angiogenesis is the process of formation of capillaries that sprout from existing blood vessels, the process includes degradation of extracellular matrix, endothelial cell proliferation, migration and differentiation. Since tumor growth and metastasis are angiogenesis-dependent, the factors that regulate angiogenesis could affect tumor growth. In 1994, Folkman and his colleagues have identified a protein which could inhibit angiogenesis of lung metastasis of a murine Lewis lung carcinoma and was named angiostatin. Angiostatin was purified from serum of mice bearing Lewis lung carcinoma. Amino acid sequence analysis reveals that angiostatin is identical to a 38 kDa internal fragment of mouse plasminogen, consisting of kringle domain 1-4(K1-4). Angiostatin specifically inhibits bFGF induced endothelial cell proliferation and tumor growth in vivo. In 1997, Cao reported that kringle 5 domain of human plasminogen is a specific inhibitor for endothelial proliferation, and kringle 5 appears to be more potent than angiostatin. We have isolated the kringle 1-5 fragment (K1-5) of human plasminogen. K1-5 could inhibit endothelial proliferation and tumor growth in vivo, and the effects appear to be greater than that of angiostatin.
To further investigate the effect of each kringle domain on angiogenesis, Pichia expression system was used to express the recombinant protein : K1-5, K2-5 and K1-3+5 . The effects of these fragments on endothelial cell proliferation were determined. Furthermore, flow cytometry was used to study the effects of K1-5 recombinant proteins on endothelial cell cycle progression.
The Pichia express vectors with insertion of genes for K1-5, K2-5, K1-3+5 were constructed. The recombinant proteins were expressed and secreted into medium; and purified by concentration and DEAE Sepharose CL-6B and lysine-Sepharose affinity chromatographies. The endothelial cell proliferation assay was performed and the result showed that all recombinant proteins could inhibit the proliferation of both bovine aortic endothelial and human microvascular endothelial cell. K1-5 is more potent than that of other fragments in inhibition of endothelial cell proliferation. The results of flow cytometry showed that recombinant proteins could inhibit endothelial cell cycle progression and induce apoptosis.
Human K1-5 and related fragments(K1-4, K1-3+5, plasminogen) were constructed in a mammalian expression vector pEGFP-N1. The recombinant plasmid was transfected into Lewis lung carcinoma (LLC) cells. The stable clones were selected and the expression of K1-5 fragments was demonstrated by dot blotting. The effects of K1-5 and related proteins to the growth of LLC were evaluated, conditioned media of transfected cells did not inhibit tumor cell growth in culture. Implantation of stable clones expressing human K1-5 and related proteins in C57BL/6 mice inhibited primary tumor growth by a range from 27% to 62%.
中文摘要2
Abstract4
致謝7
圖、附錄目錄9
儀器11
藥品13
縮寫檢索表16
緒論17
材料與方法23
結果44
討論49
參考文獻54
圖表59
附錄76
自述83
參考文獻
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