鱼精蛋白体外抑制RF/6A细胞中血管内皮生长因子表达的研究

The downregulating effect of protamine sulfate on VEGF expression and VEGF-VEGF receptor binding in vitro

目的研究鱼精蛋白对体外培养的RF/6A细胞中血管内皮生长因子(VEGF)表达的抑制作用。方法建立RF/6A细胞的正常及缺氧培养模型,在上清液中加入鱼精蛋白,质量浓度分别为10、20、40、80、160μg/mL。在细胞培养的0、24、48、72、96、120h取上清液行ELISA检测,定量分析细胞的VEGF表达情况。在培养96h时,取出细胞铺片行免疫病理学检测,检测VEGF与受体的结合状况。结果在10～80μg/mL的范围内,鱼精蛋白的抑制作用与质量浓度成正比,80μg/mL为最佳的抑制质量浓度。缺氧条件下RF/6AVEGF的表达量始终高于正常氧浓度条件下;正常和缺氧状态下,鱼精蛋白均可以明显抑制VEGF的表达,不同组的差异有统计学意义(F=7.85,P=0.002)。免疫病理染色表明鱼精蛋白减少了VEGF与其受体的结合。结论一定质量浓度的鱼精蛋白对正常及缺氧状态下的RF/6AVEGF的表达有明确的抑制作用,同时还可以减少VEGF与VEGF受体的结合。鱼精蛋白有可能成为临床治疗缺血型新生血管性眼底病的新药。

Objective A lots of researches showed that protamine sulfate,a common anticoagulation drug,inhibits the neovascularization of tumor.The aim of this study was to investigate the inhibitory effect of protamine sulfate on vascular endothelial growth factor（VEGF） expression and binding to VEGF receptor in vitro.MethodsMonkey retinal and choroidal vascular endothelial cells（RF/6A cell line） were cultured in vitro.Different concentrations of protamine sulfate（10,20,40,80,160μg/mL） were added to the culture medium in the second and third generations of cells.The supernatant of cultured cells was collected to evaluate the inhibitory effect of protamine sulfate by ELISA and immunohistochemistry at 0,24,48,72,96,120 hours respectively after addition of protamine sulfate.ResultsThe VEGF level in supernatant of cultured cells was gradually reduced with the increase of protamine sulfate concentration in the range of 10,20,40,80μg/mL with the optimum inhibitory concentration at 80μg/mL.VEGF expression level was higher in hypoxic groups than the normal control group,and a significant decrease in VEGF expression was seen between protamine treating groups and control groups in both normal and hypoxic condition（F=7.85,P=0.002）.The immunochemistry assay indicated that the VEGF expression score was 2.70±0.06,5.02±0.20,1.03±0.16,3.75±0.63 in normal control group,hypoxic control group,normoxic＋protamine group and hypoxic＋protamine group respectively,showing a statistically significant difference among them（F=43.43,P〈0.01）,between normal control group and normoxic＋protamine group（P〈0.01） or hypoxic control group and hypoxic＋protamine group（P〈0.05）.ConclusionProtamine sulfate can inhibit the VEGF expression and VEGF-VEGF receptor binding in vitro in a dosage-dependent manner at a limiting concentration range,especially under the hypoxic condition.Protamine sulfate may be used in inhibiting neovascularization in angiogenic eye diseases.