血管性血友病因子裂解酶对血管新生的抑制作用

Inhibitory effect of yon Willebrand factor-cleaving protease on angiogenesis

目的 观察血管性血友病因子裂解酶（ADAMTS13）对血管内皮细胞生长因子（VEGF）介导的血管新生的抑制作用.方法 以不同浓度的ADAMTS13（1、5、25、50、100 nmol/L）处理脐带静脉内皮细胞（HUVEC）,采用MTT法检测ADAMTS13对HUVEC增殖的影响,通过管腔形成实验观察ADAMTS13对HUVEC分化的影响,通过刮伤愈合实验观察ADAMTS 13对HUVEC迁移的影响,利用鸡胚绒毛尿囊膜实验和基质胶塞实验观察ADAMTS13在体内对血管新生的影响.结果 与对照组相比,25、50、100 nmol/L ADAMTS13对HUVEC增殖均有明显的抑制作用（P值均＜0.01）.在刮伤愈合实验中,制造损伤8h后,对照组HUVEC的迁移距离为（79±22）μm,VEGF处理组为（250±8） μm,VEGF＋ADAMTS13处理组为（170±23）μm,组间差异均有统计学意义（P值均＜0.05）.在管腔形成试验中,VEGF处理组、VEGF＋ADAMTS13处理组HUVEC培养16h后形成的管状结构长度分别是对照组的（450.6±16.6）％、（235.3±19.0）％,VEGF＋ ADAMTS13处理组管状结构少于VEGF处理组（P＜0.001）.鸡胚绒毛尿囊膜实验中,VEGF（20 ng/ml）、ADAMTS13（100 nmol/L）、ADAMTS13（100nmol/L） ＋VEGF（20 ng/ml）处理组的血管形成数量分别为对照组的（228.2±10.8）％、（69.2±21.1）％、（184.6±15.2）％.基质胶塞实验结果显示VEGF＋ADAMTS13处理组小鼠体内的血管数量为VEGF组的43.5％.结论 体外实验结果表明ADAMTS13对HUVEC增殖、分化、迁移能力均有抑制作用;体内实验结果提示ADAMTS13对血管新生有抑制作用.

Objective To investigate the inhibitory effect of von Willebrand factor-cleaving protease,a disintegrin-like and metalloprotease with thrombospondin-1 repeats （ADAMTS13） on angiogenesis induced by vascular endothelial growth factor （VEGF） in vitro and in vivo.Methods Cell proliferation assay,differentiation （tube formation） assay and wound migration assay were performed by using human umbilical vein endothelial cells （HUVECs） to explore the effect of ADAMTS13 on angiogenesis in vitro.Cells were treated with different concentrations ofADAMTS13 （1,5,25,50 and 100 nmol/L） and the number of cells was counted via MTT assay.In addition,effect of ADAMTS13 on differentiation was assessed by measuring the length of capillary-like tube structures formed by HUVECS in matrigel.Effect of ADAMTS13 on HUVEC migration was assessed via calculation of wound healing distance after 8 hrs culture with VEGF or ADAMTS13.Chick embryo chorioallantoic membrane （CAM） assay and Matrigel plug assay were performed to investigate the effect of ADAMTS13 on angiogenesis in vivo.Results ADAMTS13 significantly inhibited the proliferation of HUVECs induced by VEGF in a dose-dependent manner.Migration distance of HUVECs was （79±22） μm in control group,（250±8）μm in VEGF-treated group and （170±23）μm in VEGF and ADAMTS 13 cotreated-group after 8 hrs,respectively.The tube length is （450.6±16.6）% in VEGF-treated group and （235.3±19.0）% in VEGF and ADAMTS13 cotreatcd-group of that of control group after HUVECs cultured in matrigel for 16 hrs.The number of blood vessels decreased after treatment with ADAMTS13 in CAM assay.The number of blood vessels was （228.2± 10.8） %,（69.2±21.1） %,（184.6± 15.2）% in VEGF treated-group,ADAMTS 13 treated-group and VEGF and ADAMTS 13 cotreated-group of that of control group,respectively.Formation of capillary-like network in matrigel plugs containing VEGF was reduced to 43.5% by ADAMTS13 in matrigel plug assay in mouse model.Conclusion ADAMTS13 inhibits the HUVECs proliferation,differentiation and migration in vitro.ADAMTS13 inhibits chick embryos vascularization and formation of capillary-like network in vivo.