血管新生的過程在人類胚胎發育時期及成人期皆可進行，許多生理及病理過程亦需要血管新生作用的參與，如:組織再生、女性生理週期及傷口癒合、腫瘤生長與轉移等，血管新生作用亦扮演重要的角色。很多研究報告指出腫瘤形成的過程中，癌細胞會分泌出促血管新生的因子，刺激新增血管的生長。Angiostatin是人類血纖維蛋白溶酶原蛋白水解後的片段，為一種內生性血管新生抑制物，且會抑制內皮細胞的增生和遷移。凝血酶調節素(thrombomodulin；簡稱TM)，為一醣蛋白的接受器，TM是身體中重要的抗凝血分子，其主要功能是藉著結合凝血酶，進而限制凝血酶的促凝血活性及生理活性。

　　在本研究中我們利用酵母菌表現系統來表現並純化出kringle1-5 (K15)片段，其為angiostatin的一種形式。首先我們發現，加入 K15蛋白15小時後，牛動脈內皮細胞中的TM的量會降低，然而這個現象只有在內皮細胞中觀察到。在人類角質細胞、人類肺腺癌细胞或是穩定表現TM基因的人類黑色素瘤細胞中並沒有相同的作用。進一步研究K15降低TM細胞內含量的機制。於是利用各種蛋白質分解酶抑制劑並不會降低TM降解的結果，顯示出TM的降解不是藉由增加蛋白質分解酶活性而去切除細胞膜上的TM。另外使用吞噬作用的抑制劑，TM降解的現象則被抑制，而處理蛋白酶體抑制劑以及溶酶體抑制劑都能抑制TM減少的現象。同時，我們利用反轉錄酶-聚合酶連鎖反應偵測K15處理後TM的mRNA變化，發現到TM之mRNA不受影響。除此之外，我們若以PKA的抑制劑KT5720及H89前處理細胞， 結果發現都能抑制TM被降解的結果。

　　綜合以上的結果，我們發現K15蛋白可能是經由引發TM的內吞作用，將內皮細胞中的TM送至蛋白酶體和溶酶體進行裂解作用。不是透過活化蛋白水解酶的活性或是降低其mRNA表現所造成的現象。並且PKA可能參與在K15降低TM的過程中。

　Angiogenesis plays an important role in many physiological and pathological processes, including embryonic vascular system development, wound healing, and reproductive cycle in adult female and tumorigenesis. Many reports indicate that cancer cells can secrete angiogenic factors to stimulate the growth of new blood vessels during the formation of tumor. Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and inhibitor of endothelial cell proliferation and migration. Thrombomodulin (TM) is a glycoprotein receptor and cofactor for thrombin’s activity in a physiologically important natural anticoagulant system.

　In this study, we used Pichia pastoris expression system to prepare kringle 1-5 (K15) protein, a kind of angiostatin. First, we found that K15 downregulated TM expression in bovine aortic endothelial cells (BAECs) at 15 hours after treatment. The effect of K15 was endothelial cells-specific since other cell lines, such as A549 cells, HaCat cells, and A2058 cells transfected with full-length TM, had no such effect. To determine the mechanism of TM downregulation by K15, the protease inhibitors were used to inhibit this effect. The result showed that the downregulation of TM by K15 was caused by neither matrix metalloproteinases (MMPs) nor serine proteases. However, pretreatment with the internalization, proteasome or lysosome inhibitors, the expression of TM was not declined in BAECs by treatment with K15. Furthermore, the result of RT-PCR showed that the downregulation of TM expression was not due to decrease RNA expression. Additionally, pretreatment of protein kinase A (PKA) inhibitors, KT5720 and H89, could inhibit the TM downregulation by K15.

　Based on these results, we suggested that K15 induced downregulation of TM expression through proteasome-lysosome degradation pathway, not by induction of protease activity or downregulation of its transcription level. Moreover PKA was shown to be involved in this pathway.

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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。

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。

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