二十碳五烯酸对体外培养的人血管内皮细胞增殖抑制效应的研究

目的探讨二十碳五烯酸（EPA）对体外培养的人血管内皮细胞增殖的抑制效应及其机制。方法采用四甲基偶氮唑蓝（MTT）比色法,应用酶联免疫检测仪,测定不同浓度EPA对体外培养的人血管内皮细胞吸光度（A）值的影响。采用重复测量的实验设计类型,观察EPA对体外培养的人血管内皮细胞抑制效应的剂量和时间依赖性。应用SPSS13．0软件对数据进行统计学处理。各组间A值和细胞增殖抑制率的比较,采用单因素和重复测量设计的方差分析。应用流式细胞仪检测EPA对体外培养的人血管内皮细胞周期、增殖指数及凋亡的影响,检测结果应用R×C列联表的x^2检验进行统计学分析。结果MTT比色法显示EPA浓度≥0．15g／L时,对体外培养的人血管内皮细胞A值降低的影响有统计学意义（0．15g／L EPA组与空白组比较P=0．014,0．20g／L EPA组与空白组比较P=0．001）,并随时间的延长而增强,与浓度无关;对其细胞增殖抑制率的影响亦有统计学意义（0．15g／L EPA组与空白组比较P=0．02,0．20g／LEPA组与空白组比较P=0．001）,且随时间的延长而增强。流式细胞仪分析结果显示,用药组细胞增殖指数为23．9％,低于未用药组（26．9％）,无凋亡峰出现,表明EPA仅通过影响细胞增殖周期的变化抑制细胞增殖,无直接促进凋亡的作用。结论EPA对体外培养的人血管内皮细胞增殖具有抑制效应,该效应是通过封闭flk-1受体和影响细胞增殖周期实现;EPA对体外培养的人血管内皮细胞无直接促进凋亡的作用,因此不会对体内正常的血管内皮细胞产生损害,具有良好的临床应用前景。

The inhibition effects of EPA on the proliferation of human vascular endothelial cells

OBJECTIVE: To investigate inhibition effects and the mechanism of EPA on the proliferation of human umbilical vascular endothelial cells (HUVEC). METHODS: Different concentrations of EPA were added to the cultured HUVEC in vitro. The time cause and does response for the inhibition the cells proliferation in all groups were measured by the MTT assay. Light absorption values and cytostasis ratios in all groups were compared. One-way ANOVA in the SPSS 13.0 version statistical software was used. The effect of EPA on cell cycle, proliferative index (PI) and apoptosis of HUVEC in vitro were observed by Flow Cytometry. chi2-test of R x C contingency table was used as a method for statistical analysis. RESULTS: When the concentration of EPA was equal to or more than 0.15 g/L, MTT assay showed a significant difference of light absorption value in the cultured cell after EPA exposure compared with control, the suppressing effects enhanced as the treatment time increased. The peak time of the inhibition of the cell proliferation induced by EPA was at 60 hours and the effect was last until 72 hours. The proliferative index in the treatment group was 23.9%, which was lower than that in the control group (26.9%). No apoptosis was found in the cell in each group. CONCLUSIONS: EPA plays an important role of inhibition of proliferation of cultured HUVEC in vitro. No apoptosis was induced by the exposure HUVEC to EPA, therefore, it suggests a potential application for clinical trial.