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目的考察血小板激活因子(platelet activating factor,PAF)受体拮抗剂SY0916对巨噬细胞释放血管内皮生成相关因子的影响,探讨SY0916抗血管生成的相关分子机制。方法采用Boyden Chamber法检测小鼠腹腔巨噬细胞的趋化反应;放射性免疫分析法检测巨噬细胞U937上清中白细胞介素(IL)-1β含量;L929生物测定法分析肿瘤坏死因子(TNF)-α分泌;ELISA法测定血管内皮细胞生长因子(VEGF)含量;明胶酶谱法检测间质金属蛋白酶(MMP)-9活性;Western Blot法检测MMP-9蛋白表达;RT-PCR法观察IL-1β、TNF-α、VEGF mRNA的表达;凝胶电泳迁移率变更法(EMSA)分析核转录因子(NF)-κB活性。结果 SY0916对PAF诱导的小鼠腹腔巨噬细胞趋化反应具有显著的抑制作用;SY0916可呈剂量依赖性地抑制PMA刺激人U937巨噬细胞引起的IL-1β、TNF-α、VEGF的生成以及IL-1β、TNF-α、VEGF的mRNA表达;SY0916能显著抑制U937细胞MMP-9的活性及蛋白表达;SY0916明显抑制NF-κB的活化。结论 PAF受体拮抗剂SY0916可能通过抑制巨噬细胞NF-κB的活性而下调促血管生成因子和MMP的表达发挥抗血管生成的作用。
Objective To investigate the effect of SY0916, a platelet activating factor (PAF) receptor antagonist, on the release of vascular endothelial growth factor (RVA) in macrophages and to explore the molecular mechanism of SY0916 anti-angiogenesis. Methods The chemotactic response of peritoneal macrophages in mice was detected by Boyden Chamber method. The content of interleukin (IL) -1β in supernatant of macrophage U937 was detected by radioimmunoassay. Tumor necrosis factor (TNF) -α secretion; the content of vascular endothelial growth factor (VEGF) was measured by ELISA; the activity of matrix metalloproteinase (MMP) -9 was detected by gelatin zymography; the expression of MMP-9 was detected by Western Blot; 1β, TNF-α and VEGF mRNA were detected by enzyme linked immunosorbent assay (ELISA). The activity of nuclear factor kappa B (NF-κB) was analyzed by electrophoretic mobility shift assay (EMSA). Results SY0916 inhibited PAF-induced chemotaxis of murine peritoneal macrophages significantly. SY0916 inhibited the production of IL-1β, TNF-α and VEGF induced by PMA in human U937 macrophages in a dose-dependent manner The expression of IL-1β, TNF-α and VEGF mRNA was significantly inhibited by SY0916. The activity and protein expression of MMP-9 in U937 cells were significantly inhibited by SY0916. SY0916 significantly inhibited the activation of NF-κB. Conclusion PA09 receptor antagonist SY0916 may play an anti-angiogenic effect by inhibiting the activity of NF-κB in macrophages and down-regulating the expression of angiogenic factors and MMPs.