乙酰肝素酶抑制剂对体外培养视网膜色素上皮细胞增殖的影响

Effect of heparanase inhibitor on proliferation of retinal pigment epithelial cell

摘要:

目的：探讨乙酰肝素酶（heparanase-1, HPA-1）抑制剂应用对体外培养的视网膜色素上皮(retinal pigment epithelial, RPE)细胞增殖的影响。方法：采用DMEM培养基(Dulbecco''s modified eagles medium, DMEM)体外培养人RPE细胞,选择第5代细胞用于实验。倒置显微镜下直接观察不同质量浓度硫代磷酸甘露醇戊糖（phosphomannopentaose sulfate, PI-88）对人RPE细胞体外生长的干预效果,应用四甲基偶氮唑盐[3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny1 tetrazolium bromide, MTT]比色法检测RPE细胞A₅₇₀值；免疫组织化学方法检测人RPE细胞角蛋白和HPA-1表达。 结果：体外增生的RPE细胞胞浆和细胞核HPA-1呈强阳性表达,以细胞浆内表达为主,PI-88干预后细胞浆表达减弱。PI-88对体外RPE细胞的增生有明显的时效和量效抑制关系,药物干预72h细胞A₅₇₀值随质量浓度不同表现明显下降趋势（P<0.05）；而同一质量浓度,药物质量浓度达到100mg/L及以上时,48~72h才表现A₅₇₀值减小的趋势。结论: HPA-1抑制剂可抑制体外培养RPE细胞的增生,并呈现剂量和时间依赖关系。

Abstract:

AIM: To investigate the effect of heparanase (HPA-1) inhibitor on the proliferation of human retinal pigment epithelial (RPE) cell. METHODS:Hunan RPE cells were primarily cultured and the fifth generation cells were used in the experiment. The phosphomannopentaose sulfate(PI-88) solutions of different concentrations were added into the medium for 48 and 72 hours to co-culture the cells. The proliferation of human RPE cell was estimated using MTT colorimetric assay as the A₅₇₀ value. The expression of keratin and HPA-1 in the cells was detected by immunochemistry.RESULTS: HPA-1 was intently expressed in both the cytoplasm and nucleus of human RPE cells, and weak expression was seen in RPE cells co-cultured with PI-88. Either in 48 hours or 72 hours, the A₅₇₀ values of various concentrations of PI-88 culture group were significantly declined in comparison with without PI-88. When PI-88 was administered for 72 hours, the A570 values showed the tendency of reduction to those of 48 hours in >100mg/L groups.CONCLUSION:HPA-1 inhibitor can suppress the proliferation of human RPE cell at the concentration- and time-dependent manner in vitro.