重组人血管内皮抑制素抑制内皮细胞血管生成的实验研究

Inhibitory effect of human recombinant endostatin(endostar) on the angiogenesis of HUVECs

目的:观察国产重组人血管内皮抑制素(Endostar,恩度)对人脐静脉内皮细胞(HUVECs)的抗血管生成作用,并对其机制进行初步探讨。方法:采用MTS/PMS系统测定不同浓度恩度对HUVECs和人肝癌细胞株HepG2的生长抑制作用;流式细胞术检测恩度在相同时间内诱导HUVECs和HepG2细胞早期凋亡和晚期凋亡情况;通过迁移/侵袭实验、体外管腔形成实验和鸡胚尿囊膜(CAM)实验观察恩度对HUVECs迁移/侵袭和体内外管腔形成及功能的影响。结果:不同浓度的恩度持续作用72h,对HUVECs具有抑制增殖,且在50～200ng/ml的剂量范围内呈现浓度依赖关系;而对HepG2细胞未见明显作用。药物处理方式的不同,恩度对HUVECs诱导凋亡和迁移/侵袭作用差异显著;对HepG2细胞的迁移/侵袭未见明显影响。高剂量的恩度对HUVECs体外管腔形成和鸡胚尿囊膜的血管发生及成熟具有显著影响。结论:重组人血管内皮抑制素(恩度)能够有效地抑制血管内皮细胞形成复杂的管腔,并显著影响血管的成熟;对人肝癌细胞系HepG2生长及迁移/侵袭未见明显影响。

Objective：To assess the effect of human recombinant endostatin（endostar） alone on the human umbilical vein endothelial cells （HUVECs） proliferation and cord formation in vitro and to study the mechanism of antiangiogenesis. Methods：Cell proliferation in different concentration was determined by MTS/PMS（ methy thiazolyl tetrazolium, inner salt/phenazine methosulfate） assay. The fluorescence flow cytometry（FCM） Annexin V-FITC assay was applied to detect the changes of apoptotic rate at early and late apoptosis process. The effect of endostar on HUVECs migration/invasion, angiogenesis and the subsequent growth of the angiogenic vessels were observed by transwell inserts tests in vivo, tube formation tests and the chick chorioallantoic membrane （CAM） assay. Resuits：After incubation of HUVECs with vary doses of endostar for 72 hours,the inhibited effect was observed. However, we found the inhibited effect of endostar alone was dose-dependent from 50ng/ml to 200ng/ml. The effect of endostar was also observed for inhibition of human umbilical vein endothelial cells＇ proliferation and migration in vitro. Endostar treatment group showed shorter length of blood vessels and smaller area of blood vessels than control group. Furthermore,experiments of inhibition of proliferation performed on nonmicroendothelial cells showed that endostar was ineffective. Conclusion：These data strongly indicates that eudostar doesn＇t show any effects on the human lepatic carcinoma cell line HepG2, but it is a potent angiogenesis inhibitor and seriously effects the integrity of the blood vessels.