特异性血管生成抑制素的制备及其抑制血管生成作用

Preparation of native angiostatin from human plasma and its antiangiogenesis effects

目的： 从血浆中纯化制备有特异抑制血管生成活性的天然ａｎｇｉｏｓｔａｔｉｎ 蛋白，为基础研究和应用研究打下基础．方法： 制备Ｌ－ｌｙｓｉｎｅ 偶联的Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析柱，从血浆中纯化纤溶酶原，用弹性蛋白酶在体外进行有限消化，再以Ｌ－ｌｙｓｉｎｅ Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析纯化获得天然ａｎｇｉｏｓｔａｔｉｎ 片段，通过体外血管内皮细胞生长抑制分析和体内鸡胚尿囊膜血管生成抑制实验测定纯化蛋白的活性． 结果： 以Ｌ－ｌｙｓｉｎｅＳｅｐｈａｒｏｓｅ ４Ｂ从血浆中纯化得到单一成分的纤溶酶原蛋白，弹性蛋白酶有限消化后进一步的亲和层析纯化得到ａｎｇｉｏ－ｓｔａｔｉｎ 蛋白；该蛋白体外可抑制牛主动脉内皮细胞的生长，体内可抑制鸡胚尿囊膜血管生成． 结论： 该实验方法可纯化得到有活性的天然ａｎｇｉｏｓｔａｔｉｎ 蛋白．

AIM: To develop a method to prepare native angiogenesis inhibitor angiostatin from human plasma for research and practical use. METHODS: L lysine linked Sepharose 4B was prepared by ligation of L lysine to CNBractivated Sepharose 4B. Affinity chromatograph with L lysine Sepharose 4B was utilized to purify plasminogen from human plasma and limited proteolysis by Elastase was applied to purified plasminogen. Angiostatin fragments were purified by using L lysine Sepharose 4B. BAE cell growth inhibition assay and CAM angiogenesis assay were carried out to check the activity of the purified angiostatin. RESULTS: Plasminogen was purified though affinity chromatograph with L lysine Sepharose 4B. Angiostatin was generated though limited proteolysis by Elastase and purified by affinity chromatograph with L lysine Sepharose 4B. Angiostatin had potent inhibitory effects on bovine aortic endothelial cell growth and CAM angiogenesis. CONCLUSION: Active native angiostatin is purified by our method.