地鳖虫纤溶活性蛋白组分抑制血管的生成

Antiangiogenesis of fibrinolyric protein from Eupolyphaga sinesis Walker

从地鳖虫(Eupolyphaga sinesisWalke)中分离纯化出纤溶活性蛋白组分———地鳖虫纤溶活性蛋白(Fibrinolyric protein ofEupolyphage sinesis,EFP),研究其对血管生成的抑制作用。以新鲜地鳖虫为原料,采用硫酸铵分段盐析,经DEAE-纤维素和SephadexG-75柱等方法分离纯化EFP,用不同浓度的EFP作用于人微血管内皮细胞(MVEC),用MTT法测定EFP对MVEC增殖的影响,用单细胞凝胶电泳和AnnexinV-FITC/PI双荧光染色观察细胞凋亡情况,采用流式细胞术检测EFP对MVEC细胞周期的影响,并采用鸡胚绒毛尿囊膜的新生血管模拟肿瘤的新生血管,检测EFP对鸡胚尿囊膜(CAM)血管生成的作用。结果表明:1)MTT法测得EFP可抑制MVEC细胞增殖,增殖抑制率达46%;2)EFP可诱导MVEC细胞凋亡,且成剂量依赖关系,用单细胞凝胶电泳法检测细胞凋亡,随着EFP浓度的增大,拖尾细胞增多;用AnnexinV-FITC/PI进行双荧光染色发现,当EFP处理浓度达2μg/ml时,出现了大量凋亡晚期细胞;3)EFP可干扰MVEC的细胞周期,出现S期和G2/M期阻滞;4)用鸡胚尿囊膜实验观察到EFP剂量为0.06mg/egg即可抑制鸡胚尿囊膜血管生成,最高抑制率可达35.8%。提示地鳖虫纤溶活性蛋白组分具有抑制血管生成的作用。

A kind of fibrinolytic protein was separated and purified from Eupolyphaga sinensis Walke, and used to investigate the anti-angiogenic effect of EFP in the paper. EFP was purified by ammonium sulphate precipitation, DEAE-cellulose and SephadexG-75 coIumn chromatography from fresh Eupolyphaga sinesis Walker. Human microvascular endothelial cells （MVEC） were used to test the anti-angiogenic effect of EFP. MVEC proliferation was detected by MTT assay; and their apoptosis was observed by single-cell gel electrophoresis assay （SCGE） and Annexin V-FITC/PI double fluorescence dyeing. The effects of EFP on cell cycles of MVEC was tested by flow cytometry analyses, and chick embryo assay was used to test the inhibitory effects of EFP on neovascularization. Results showed that： 1） EFP inhibited the proliferation of MVEC, and their growth inhibition rate reached to 46% ; 2） EFP induced apoptosis of MVEC, and the degree of apoptosis of the cells and the level of DNA strand breaks were dependent on the EFP concentration. Observed with Annexin V-FITC/PI double fluorescence dyeing, when EFP concentration reached 2 μg/ml, a great many of terminal apoptosic cells were appeared; （3） EFP could cause cell cycle arrest at S and G2/M phases; 4） the vascular network of chorioallantoic membranes （CAM） of chicken embryo was inhibited in 0.06 mg/egg EFP group, and the highest inhibition was 35.8%. These results indicate that EFP has anti-angiogenic activity.