| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 91 === 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 K15K4(kringle 4 deleted). In proliferation assay, K25 exhibited potency in inhibition of BAECs proliferation. In migration assay, K15K4 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 K15K4 attenuated bFGF-induced capillary density and hemoglobin content in subcutaneously implanted Matrigel plugs in mice. Systemic administration of K15、K25 and K15K4（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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