血管新生(angiogenesis)是由存在的血管中生長出新血管的過程，此過程於許多生理及病理的變化中扮演重要的角色，如胚胎的發育、女性生殖週期的子宮內膜增生、傷口癒合及腫瘤的轉移等。許多研究報告指出腫瘤在形成的過程中，腫瘤細胞會釋放出血管新生因子，促進在腫瘤中或周圍產生血管新生作用，藉以新生血管提供腫瘤生長代謝所需要的物質。血管靜止蛋白（angiostatin），為血纖維蛋白溶酶原(plasminogen)的蛋白質片段，包含kringle 1-4 domain (K14)的區域，具有抑制血管新生的能力。最近的研究結果指出，由利用酵素水解出的人類血纖維蛋白溶酶原片段kringle 1-5 domain(K15)及kringle 5，比血管靜止蛋白（angiostatin）具有更好的抑制血管新生的效果。許多研究顯示出，不同來源的kringle片段具有不同抑制血管新生的效果，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，我們利用酵母菌表現系統表現重組K15、K25 (去除kringle 1區域)和K15K4(去除kringle 4區域)。將表現及經純化後之重組蛋白進行抑制內皮細胞生長及移動之分析發現，K25在抑制內皮細胞生長的實驗中具較佳的抑制效果，而K15K4則在抑制內皮細胞移動的實驗中，有較明顯的抑制效果。我們進一步在活體(in vivo)的狀態下，分別利用Matrigel plug和蛋白直接注射法抑制腫瘤生長實驗來研究這些不同重組蛋白在活體內抑制血管新生作用或抑制血管新生依賴性的腫瘤生長上的作用效果。而結果顯示K15、K25和K15K4皆會使殖入在小鼠皮下的Matrigel中之血管密度和血紅素的含量降低；直接注射K15、K25和K15K4 （2 mg/kg/24 hr）蛋白質至有Lewis lung carcinoma的小鼠中，發現可使得腫瘤明顯減小，而由組織切片發現抑制腫瘤生長是由於其血管新生作用被抑制所致。由以上的實驗結果，我們可以下一個結論，當去除kringle 1或kringle 4皆會增進抑制血管新生的能力，而其中不論in vitro或in vivo皆顯示K25的效果較K15更好。
此外，為了探討人類血纖維蛋白溶脢原之kringle結構區在血管新生作用上之角色，我們將人類plasminogen及 K15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，以確認得到持續表現plasminogen及K15蛋白質的LLC細胞株。在培養皿中生長速度的觀察中發現這些表現蛋白質的LLC細胞株與LLC的生長速率相同，持續表現plasminogen及K15蛋白質的LLC細胞株並未影響細胞的生長速率。接著將穩定表現plasminogen及K15之腫瘤細胞以皮下注射方式植入小鼠體內，比較各細胞株在腫瘤生長上差異，發現表現plasminogen及K15腫瘤的生長速度較控制組(LLC)慢且體積較小，且其抑制腫瘤之能力分別為K15（58.9 %）及plasminogen（73.7 %）。因此進一步的以抗CD31單株抗體來進行組織免疫染色，也可以發現分泌plasminogen及 K15的腫瘤中的內皮細胞數量確實較控制組來的少，證明在表現血管靜止蛋白的腫瘤中的確會抑制腫瘤血管新生的情形。

Angiogenesis, plays an essential role in many physiological and pathological processes, including embryonic development, menstruation cycle, wound repair, and tumor metastasis. Many reports indicate that cancer cells can secrete angiogenic factors to induce the growth of new blood vessels during the formation of tumor. Angiostatin, containing the kringle 1-4 domain of plasminogen, is the angiogenesis inhibitor which was identified from the serum and urine of the tumor bearing mice. In the previous study we demonstrated that kringle 1-5 domain was more potent than angiostatin.

In order to improve the anti-angiogenic potency of the kringle domains of plasminogen, different kringle 1-5 fragments were prepared using Pichia pastoris expression system. We have obtained K15、K25 (deleted the kringle 1 domain)and K15K4(kringle 4 deleted). In proliferation assay, K25 exhibited potency in inhibition of BAECs proliferation. In migration assay, K15K4 displayed a significant inhibitory ability than K15 and K25 in inhibition of endothelial migration. To demonstrate that anti-angiogenic ability of these kringle fragments, Matrigel plug and a systemic administration in mice bearing Lewis lung carcinoma were performed. K15、K25 and K15K4 attenuated bFGF-induced capillary density and hemoglobin content in subcutaneously implanted Matrigel plugs in mice. Systemic administration of K15、K25 and K15K4（2 mg/kg/24 hr）in mice bearing Lewis lung carcinoma caused a reduction in tumor size, and histological analysis of tumor showed reduced numbers of endothelial cells. These data indicate that K25 displays a more potent effect on suppression of endothelial cell proliferation, angiogenesis, and tumor growth than K15.

To further investigate the role of kringle domains of plasminogen in angiogenesis, we prepared the recombinant plasmid, containing plasminogen or K15,and was transfected into Lewis lung carcinoma. Stably expressed LLC cells were implanted to C57/B6 mice and evaluated the effect of constituted expression of plasminogen or K15. The growth rate of the tumors induced by the transfected tumor cells was significantly slower than the nontransfected tumor ( 58.9% and 73.7 % slower than the control). Immunocytochemical staining of tumor sections by anti-CD31 antibody showed significant reduction of endothelial cell numbers in the plasminogen- or K15- transfected LLC cells,demonstrating that angiostatin had anti-angiogentic activity.

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