血管新生(Angiogenesis)是指從已存在的血管新生出微血管的複雜過程，發生在胚胎發育、組織修補及女性生殖週期。由於腫瘤的生長和轉移必須藉助於血管新生，因此抑制血管新生是治療癌症的新策略。血纖維靜止蛋白(Angiostatin)是血纖維蛋白溶酶原(plasminogen)的一個蛋白片段，包含前四個kringle，它是一種能抑制血管新生的內生性蛋白。本實驗室之前研究發現血纖維蛋白溶酶原片段蛋白K4430對內皮細胞的移動和增生較血纖維靜止蛋白有顯著的抑制效果。然而對於血纖維靜止蛋白抑制血管新生的機制仍眾說紛紜，因此本實驗的目的是進一步的探討血纖維蛋白溶酶原片段蛋白K4430抑制血管新生的機制。首先，血纖維蛋白溶酶原片段蛋白K4430由酵母菌系統表現再經由陰離子交換樹脂(DEAE)與親核性樹脂(lysine sepharose)純化。以血纖維蛋白溶酶原片段蛋白K4430處理牛肺動脈內皮細胞(CPAE)發現片段蛋白具有引起細胞凋亡的功能，而且呈現劑量效應。另一方面，血纖維蛋白溶酶原片段蛋白K4430可以起細胞內鈣離子濃度增加，使用鈣離子螯合劑可以抑制片段蛋白所引起的細胞凋亡及降低片段蛋白所引起的鈣離子濃度改變，也可以抑制caspase 9之活化。此外，血纖維蛋白溶酶原片段蛋白K4430造成粒線體和內質網的鈣離子濃度下降，但是使用抑制粒線體鈣離子釋放之抑制劑無法抑制片段蛋白所引起的細胞凋亡，而使用抑制內質網鈣離子釋放之抑制劑發現有些微的抑制效果。因此我們發現血纖維蛋白溶酶原片段蛋白K4430造成內皮細胞凋亡可能是透過增加細胞內鈣離子濃度而造成。

Angiogenesis is a biological process by which new capillaries are formed from preexisting vessels. It is essential for embryonic development, tissue growth and female reproductive cycle. Since tumor growth and metastasis are angiogenesis-dependent, several angiogenesis inhibitors are used as a new strategy for cancer therapy. Angiostatin, the first four kringles of plasminogen, is a potent endogenous inhibitor of angiogenesis. Previous studies in our laboratory demonstrated that the plasminogen fragment K4430 was more potent than angiostatin in suppressing endothelial cells migration and proliferation. The purpose of this study was to further clarify its mode of action to inhibit angiogenesis. Recombinant plasminogen fragment, K4430, was expressed in Pichia pastoris expression system and purified by anion exchange (DEAE) and lysine sepharose. Recombinant K4430 could induce endothelial cells apoptosis dose-dependently. Moreover, recombinant K4430 could elevate endothelial cell intracellular Ca2+ levels. The intracellular Ca2+ chelator, BAPTA-AM could inhibit recombinant K4430-induced apoptosis, the rise of intracellular Ca2+ levels and the activation of caspase 9. Besides, recombinant K4430 caused a decrease of mitochondrial Ca2+ and endoplasmic reticular Ca2+ stores. However, the pretreatment of mitochondria calcium release blocker could not inhibit recombinant K4430-induced apoptosis. And, the calcium release blockers of endoplasmic reticulum have a slight tendency to inhibit the recombinant K4430-induced apoptosis. These results suggest that recombinant K4430-induced endothelial cell apoptosis was a consequence of increase in intracellular Ca2+ levels.

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