二十碳五烯酸抑制血管内皮生长因子诱导人血管内皮细胞增生作用的研究

Inhibitory effect of eicosapentaenoic acid on proliferation of human vascular endothelial cells induced by vascular endothelial growth factor

背景研究表明二十碳五烯酸（EPA）作为主要的脂质参与人体的生物代谢活动，抑制血管内皮生长因子（VEGF）的产生、阻滞血管平滑肌细胞的内移和增生。而眼部多种疾病与新生血管的形成有关。目的探讨EPA对VEGF诱导的人脐静脉内皮细胞（UVEC）增生的抑制作用。方法对人UVEC株培养和传代，并用Ⅷ因子相关抗原多克降抗体进行免疫细胞化学鉴定。应用免疫组织化学法检测EPA对VEGF受体2（Flk-1）在人UVEC中表达的影响；用MTT比色法检测不同质量浓度EPA作用不同时间后对非VEGF诱导及VEGF诱导的人UVEC增生的抑制率；用流式细胞术检测EPA对VEGF诱导的人UVEC细胞周期的影响。结果培养和传代的人UVEC呈纺锤形及鹅卵石样排列，Ⅷ因子相关抗原鉴定呈阳性反应。EPA作用后，Flk-1在人UVEC中的阳性染色强度明显弱于对照组，Flk-1阳性细胞数明显减少。6个质量浓度组的EPA对VEGF诱导及非VEGF诱导的人UVEC的增生均有明显的抑制作用，其作用呈质量浓度依赖性（F：23．072，P=0．000）；各质量浓度组的EPA对VECF诱导的人UVEC的抑制作用强于非VEGF诱导的人UVEC，差异有统计学意义（F=41．417，P=0．000）。各质量浓度组EPA对VEGF诱导的人UVEC作用24、48、72h后，其细胞数逐渐降低（F=87．823，P=0．000），但作用时间对其抑制作用无明显影响（F=1．495，P=0．236）。EPA使VEGF诱导的人UVEC细胞阻滞在G0／G1期，EPA组G0／G1期细胞数比例为（75．83±1．56）％，对照组为（68．62±1．44）％，差异有统计学意义（t=-5．88，P=0．00）；EPA组与对照组均未发现凋亡细胞。结论EPA能够通过阻止人血管内皮细胞的DNA合成而明显抑制VEGF诱导的人UVEC的增生，其抑制作用呈剂量依赖性；EPA作用后人UVEC中Flk-1的表达下调，提示EPA可能有抗血管生成的作用。

Background Eieosapentaenoie acid （EPA） funetion as the critical lipid mediators involved in several biological events in human body and play important role in suppressing the genesis of vascular endothelial growth factor （VEGF） ,migration and proliferation of vascular endothelial cells. Many ocular diseases were proved to be associated with neovaseularization. Objective The purpose of this study was to investigate the inhibitory effect of EPA on the proliferation of human umbilical vein endothelial cells （HUVEC） induced by VEGF. Methods HUVEC strain was cultured and passaged, and different concentrations of EPA were added to the medium with and without VEGF. The cultured cells were identified by antiofactor V~ polyclonal antibody. The suppressing role of different concentrations of EPA on the proliferation of VEGF-induced or -unindueed HUVEC was assessed by MTT method. The influence of different concentrations of EPA on the cellular cycle of VEGF-induced HUVEC was assayed using flow cytometry. The expression of Flk-1, a receptor of VEGF, in the HUVEC was detected by immunohistochemistry. Results Cultured HUVEC showed the fusiform in shape and presented with the cobblestone-like arrangement with the positive response for Ⅷ factor-related antigen. Various concentrations of EPA showed obviously inhibitory effect on VEGF-induced or -uninduced HUVEC at a dose-dependent manner （F = 23. 072,P= 0. 000）. The inhibitory ability of EPA on VEGF-induced HUVEC was stronger than VEGF-uninduced HUVEC （F= 41. 417,P = 0. 000）. In 24,48 and 72 hours, the action of EPA on the proliferation of HUVEC was gradually enhanced with the prolong of time （F-= 1. 495,P = 0. 236）. Cell cycle analysis indicated that EPA arrested VEGF-induced HUVEC in G0/G1 phase. The ratio of HUVEC in G0/G1 phase in EPA group was （75.83±1.56）% , and that in control groups was （ 68.62± 1.44 ） % , showing a significant difference between them （ t = - 5.88, P = 0.00 ） , and no apoptosis of HUVEC was found in both groups. Flk-1 was strongly expressed in the cellular nucleus and cytoplasm in control group. However, the positive expressing intensity of Flk-1 in the HUVEC weakened, and the positive cell number was evidently less in EPA group. Conclusion EPA can inhibit the proliferation of VEGF- induced HUVEC through arresting the synthesis of DNA of HUVEC and downregulate the expression of Flk-1 in HUVEC. These results suggest that EPA might exert an antiangiogenic effect.