血管新生作用是由許多個步驟所組成，其中包括了內皮細胞的增生與遷移、細胞間質的分解、及長出新的血管。腫瘤的生長與轉移的過程需要血管新生的參與，因此藉由調控血管新生作用即可能影響腫瘤的生長。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的蛋白質存在，經過純化分離，由胺基酸序列分析發現其為老鼠血纖維蛋白溶酶原(plasminogen)之片段，分子量為38 kDa，此片段包含了kringle 1-4 domain，將之命名為angiostatin。Angiostatin是一種內生性血管新生抑制物，而血纖維蛋白溶酶原的kringle 5與前四個kringle具有高度的序列相似性，研究指出可以抑制血管新生。這些kringle 片段蛋白質皆可以專一地抑制內皮細胞的增生與遷移。其他的研究報告亦指出，不同的kringle片段結構在抑制內皮細胞的增生與遷移上具有不同程度的功效。為進ㄧ步研究血纖維蛋白溶酶原kringle結構在抑制血管新生上的角色，我們製備了一系列的GST-kringle融合蛋白。這些重組的GST-kringle融合蛋白以大腸桿菌表現。最後，以glutathione-Sepharose 及 lysine-Sepharose兩種親和性色層分析法純化得到kringle 1及kringle 4。有報告指出不同來源的kringle片段有不同抑制血管新生的差異。為探討kringle 1及kringle 4在抑制內皮細胞遷移活性上的差異是否會受表現系統影響，我們以細胞刮傷測試(scratch wound assay)進行抑制bFGF所引起的牛動脈內皮細胞（bovine aortic endothelial cell, BAEC）遷移之功能分析。結果發現以大腸桿菌表現之kringle1及kringle4，其抑制內皮細胞遷移效力仍是kringle 4大於kringle 1。細胞週期的控制對於細胞的增生、分化與死亡是重要的。為了研究kringle 1及kringle 4是否透過影響細胞週期的進行，而具有抑制內皮細胞增生的效果，我們以流式細胞儀進行了細胞週期的分析。結果發現kringle 1及kringle 4可以使內皮細胞的細胞週期停留在G0/G1期。由此結果，我們推測單一片段的血纖維蛋白溶酶原kringle 蛋白可能藉由調節內皮細胞之細胞週期而抑制血管新生。另外，先前實驗室已初步證實由Pichia pastoris表現之kringle 2-5 N289A/T346A/L532R為本實驗室發現在試管內最有效抑制血管新生之kringle相關蛋白。為證實其效力，在動物實驗上投予小鼠全身kringle 2-5 N289A/T346A/L532R，結果發現的確最有效地阻斷所植入Lewis lung carcinoma的生長。

Angiogenesis is a multi-step process that includes endothelial cell proliferation, migration, degradation of extracellular matrix, and new lumen organization. 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. Angiostatin is one of potent endogenous angiogenesis inhibitors. Kringle 5 domain of plasminogen, which shares high sequence homology with the four kringles of angiostatin, is also an angiogenesis inhibitor. Many reports indicate that different kringle domains have different activities on inhibition of endothelial cell proliferation and migration. In order to investigate the role of individual kringle domain of plasminogen in anti-angiogenesis, we prepared a series of GST-kringle fusion proteins. The recombinant GST-kringle fusion proteins were expressed in E. coli. Finally, kringle 1 and kringle 4 were purified by affinity chromatographies of glutathione-Sepharose and lysine-Sepharose. Provious reports indicated that kringle domains prepared by different methods have different activities on anti- angiogenesis. In order to investigate whether the different preparation methods affect the anti-migration activities of kringle 1 and kringle 4, we performed the scratch wound assay to analyze their activities on inhibition of bovine aortic endothelial cell migration. The results showed that kringle 4 had more anti- migration potency than kringle 1. The cell cycle control is very important in regulating cell proliferation, differentiation, and cell death. In order to define whether the anti-proliferation effect of kringle 1 and kringle 4 is caused by affecting endothelial cell cycle progression, we performed the cell cycle FACS analysis. The results showed that kringle 1 and kringle 4 caused endothelial cell cycle arrest in G0/G1. These results suggest that single kringle domain of plasminogen can cause endothelial cell cycle arrest. In our previous studies, kringle 2-5 N289A/T346A/L532R was the most potent antiangiogenesis agent in the in vivo assay. In order to confirm the activity of kringle 2-5 N289A/T346A/L532R in anti-angiogenesis in vivo, we performed the animal experiment. Systemic administration of the kringle 2-5 N289A/T346A/L532R had the highest activity in inhibiting the growth of inoculated Lewis lung carcinoma.

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吳柏義 (2002) 人類血纖維蛋白溶酶原之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
林忠義 (2003) 人類血纖維蛋白溶酶原之Kringle 1-5區域突變株於血管新生特性之研究，國立成功大學生物化學研究所碩士論文
張智維 (2003) 人類血纖維蛋白溶酶原之Kringle結構區對血管新生之功能分析，國立成功大學生物化學研究所碩士論文
楊晉姿 (2001) 重組人類血纖維蛋白溶酶原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