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目的:探讨人血管抑素(angiostatin)对不同细胞体外增殖的影响。方法:用亲和层析、分子筛技术及有限酶解法从人血浆组分Ⅲ中分离纯化得到人血管抑素(human angiostatin,以下简称angiostatin),采用MTT法分别分析an-giostatin作用24 h、48 h、72 h原代培养的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)、不同剂量angiostatin(20 nmol/L,40 nmol/L,80 nmol/L,160 nmol/L,320 nmol/L及640 nmol/L)作用下HUVEC和小鼠肺动脉内皮细胞(1H11)、angiostatin为160 nmol/L时其他细胞(7721、Hela、A431、A549、H128、K562、LLC、SL7、3T3)体外增殖的抑制率;透射电镜下观察angiostatin作用后1H11细胞形态的变化。结果:纯化的angiostatin能特异地抑制HUVEC细胞的体外增殖,且呈现一定的剂量和时间依赖性,HUVEC对angiostatin的敏感性高于1H11(P<0.05);对其他细胞株的体外增殖无影响;透射电镜可见1H11内皮细胞出现典型的凋亡的形态学改变。结论:人血浆组分Ⅲ来源的an-giostatin能特异地显著抑制内皮细胞的体外增殖,其作用机制之一是诱导内皮细胞凋亡。
Objective: To investigate the effect of angiostatin on the proliferation of different cells in vitro. Methods: Human angiostatin was isolated and purified from human plasma fraction Ⅲ by affinity chromatography, molecular sieve technique and limited enzymolysis method. The effects of an-giostatin on the expression of angiostatin were analyzed by MTT assay for 24 h, 48 h, 72 h of primary cultured human umbilical vein endothelial cells (HUVEC), different doses of angiostatin (20 nmol / L, 40 nmol / L, 80 nmol / L, 160 nmol / L, 320 nmol / In vitro proliferation of HUVECs and mouse pulmonary artery endothelial cells (1H11) and angiostatin at 160 nmol / L in vitro (7721, Hela, A431, A549, H128, K562, LLC, SL7 and 3T3) The morphological changes of 1H11 cells were observed under transmission electron microscope after angiostatin treatment. Results: The purified angiostatin could inhibit the proliferation of HUVEC cells in vitro in a dose and time dependent manner. The sensitivity of HUVEC to angiostatin was higher than that of 1H11 (P <0.05), but not to other cell lines. The morphological changes of typical apoptotic cells in 1H11 endothelial cells were observed by transmission electron microscopy. CONCLUSION: An-giostatin derived from human plasma fraction Ⅲ can significantly and specifically inhibit the proliferation of endothelial cells in vitro. One of the mechanisms is that it induces endothelial cell apoptosis.