Background Experimental study showed that heparanase (HPA)is overexpressed in choroidal neovascularization,suggesting that it may play a role in the pathogenesis of angiogenesis.To certify HPA inhibitor suppress the formation and development of new blood vessel has an important significance for the treatment of choroidal neovascularization.Objective The aim of this study was to investigate the effect of HPA inhibitor on the proliferation of human umbilical vein vascular endothelial cell (UVEC) and the expression of HPA.Methods Hunan UVEC was primarily cultured and passaged and the third generation cells were used in the experiment.Phosphomannopentaose sulfate (PI-88) solution,a HPA inhibitor,was prepared with endothelial cell medium and the end concentrations were 400.00,200.00,100.00,50.00,25.00,12.50,6.25 mg/L respectively.The cells were treated with PI-88 solutions for 24,48 and 72 hours.The growth and proliferation of human UVEC were analyzed using MTT colorimetric assay at absorbance 570 nm.The expression of HPA in the cells was detected by immunochemistry in 48 hours after addition of PI-88.Results Cultured human UVEC showed the fusiform and polygon in shape.The A570 values of human CVEC were significantly different among various concentrations of PI-88 groups (F=2.721,P=0.053 ) and different action time (F=9.656,P =0.002).When PI-88 was administered for 24 hours,the A570 values of human UVEC were insignificantly altered in comparison with the one without PI-88 culture group (P＞0.05 ).However,in 72 hours after experiment,the A570 values were significantly declined as the PI-88 concentration was ＞50.00 mg/L ( P＜0.05 ).When PI-88 was administered for 48 and 72 hours,the A570 values of human UVEC were significantly higher than those of 24 hours in ＜50.00 mg/L groups (P＜0.01 ),but no statistical differences were seen in ＞100.00 mg/L groups among various time points (P＞0.05 ).HPA was intently expressed in the cytoplasm of human UVEC.However,at 48 hours after addition of ＞25.00 mg/L PI-88,the HPA expression was obviously weaker.Conclusions PI-88 can suppress the growth and proliferation of human UVEC at the dose-and time-dependent manner by downregulating the expression of HPA in the cells.%背景 已有动物研究表明,乙酰肝素酶(HPA)在脉络膜新生血管(CNV)中呈高表达,提示其与新生血管的形成密切相关.确定HPA抑制剂是否可影响血管的生成和发展对研究CNV的治疗有重要意义.目的 探讨HPA抑制剂硫代磷酸甘露醇戊糖(PI-88)对HPA的表达及体外培养的人脐静脉内皮细胞(UVEC)增生的作用.方法 人UVEC原代细胞株采用内皮细胞培养基(ECM)进行培养和传代,第3～5代细胞用于实验.用ECM配制PI-88溶液,使其终质量浓度为400.00、200.00、100.00、50.00、25.00、12.50、6.25 mg/L,分别加入内皮细胞培养基培养24、48、72 h后,用四甲基偶氮唑盐(MTT)比色法检测细胞的吸光度(A570)值；并采用免疫组织化学法观察不同质量浓度PI-88作用48 h后细胞中HPA的表达.结果 正常培养的第3代人UVEC呈梭形或多角形.相同质量浓度PI-88作用不同时间培养的人UVEC A570值差异有统计学意义(F=9.656,P=0.002),不同质量浓度PI-88作用相同时间培养的人UVEC A570值差异无统计学意义(F=2.721,P=0.053).PI-88作用24 h,各质量浓度PI-88组与0 mg/L PI-88组UVEC A570值的差异均无统计学意义(P＞0.05),当PI-88的质量浓度＞50.00 mg/L时,培养72 h的人UVEC A570值均明显低于0 mg/L PI-88组,差异均有统计学意义(P＜0.05).PI-88作用延长至48 h和72 h时,在＜50.00 mg/L PI-88组人UVEC A570值均明显高于作用24 h时,差异均有统计学意义(P＜0.05)；但各时间点≥100.00 mg/L PI-88组人UVEC A570值差异均无统计学意义(P＞0.05).HPA在生长旺盛的UVEC细胞质内呈强阳性表达,少数细胞核也有表达；不同质量浓度PI-88溶液干预48 h,25.00 mg/L PI-88组人UVEC中HPA在细胞质内表达明显减弱.结论 PI-88能够抑制人UVEC的增生,尤其是当PI-88的质量浓度＞100.00 mg/L,其对人UVEC增生的抑制作用呈剂量和时间依赖性,其作用机制主要是下调HPA在人UVEC中的表达.
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