TSP-1和NF-κB信号通路在普萘洛尔对血管瘤内皮细胞抑制机制中的作用

目的 探讨凝血酶敏感蛋白-1 (thrombospondin-1,TSP-1)和核因子κB(nuclear factorkappa B,NF-κB)信号通路在普萘洛尔对血管瘤内皮细胞抑制机制中的作用,完善普萘洛尔治疗血管瘤机制,为其临床治疗提供新靶点.方法 手术获取增生期血管瘤标本,孵育消化制成细胞悬液,流式细胞仪分选出CD31阳性血管瘤内皮细胞传代培养.加入不同浓度普萘洛尔(0、25、50、100、150、200、250μmol/L)溶液,培养24 h、48 h及72 h后,分别加入CCK-8培养液和BrdU标记液,酶标仪检测450nm处吸光度判定细胞活性和增殖情况;Westem blot检测普萘洛尔干预前后TSP-1及NF-κB通路蛋白表达情况;细胞荧光双标后,激光共聚焦显微镜观察干预前后TSP-1和NF-κB通路激活失活情况.所有数据应用SPSS 13.0统计软件进行分析.结果 流式细胞仪分选CD31阳性细胞率达98.0％.加入普萘洛尔后,随着培养时间延长,细胞活性和增殖状态受抑越明显.普萘洛尔浓度达100～150μmol/L时,细胞活性和增殖开始明显受抑,和对照组相比差异有统计学意义(P＜0.05),以72 h时为著;TSP-1及其受体CD36表达量随着普萘洛尔浓度的增加逐渐增多,而NF-κ Bp65及p-IκBα、p-IKKβ表达量开始逐渐减少(P＜0.05).两个通路存在负向调控,TSP-1逐渐激活时,NF-κBp65逐渐失活.结论 普萘洛尔可能通过促进TSP-1诱导的血管生成抑制和/或阻断NF-κB介导的促血管生成来治疗婴幼儿血管瘤,两个通路存在负向调控作用.

Roles of thrombospondin-1 and nuclear factor-kappa B signal pathways in propranolol inhibiting hemangioma-derived endothelial cells

Objective To explore the roles of thrombospondin-1 (TSP-1) and nuclear factorkappa B (NF-κB) signal pathways in propranolol inhibiting hemangioma-derived endothelial cells (HemECs) to improve the therapeutic mechanism of propranolol and provide new targets for its clinical treatment.Methods The specimens of proliferative hemangioma were obtained operatively.Cell suspension was prepared after incubation and digestion.Then CD31-positive HemECs were sorted out by flow cytometry and sub-cultured.Propranolol in different concentrations (0,25,50,100,150,200,250μmol/L) was added into HemECs solution and cultured for 24,48 and 72h respectively.And CCK-8 culture and BrdU labeling solutions were added.Activity and proliferation of HemECs before and after intervention of propranolol were respectively tested by enzyme-linked imunosorbent assay (ELISA) and protein expressions of TSP-1 and nf-kappa B pathways detected by Western blot.After immunofluorescent double-labeling,activation of TSP-1 pathway and inactivation of nf-kappa B pathway were simultaneously observed by laser confocal microscope.All data were analyzed by SPSS 13.0 statistical software.Results The rate of CD31-positive cells was 98.0％.The longer incubation time,the more marked inhibitions of cell activity and proliferation after propranolol dosing.The inhibition of cell activity and proliferation were significant at a propranolol concentration of 100-150 μmol/L.And significant differences existed between them and those of control group (P＜0.05),especially at 72 h.Expressions of TSP-1 and its receptor CD36 increased with rising propranolol concentrations while the expressions of nf-kappa Bp65,p-IκBα and p-IKKβ gradually decreased (P＜ 0.05).Two pathways were subject to negative regulations,that is,TSP-1 was gradually activated while nf-kappaBp65 became gradually inactivated.Conclusions The effective treatment of propranolol for infantile hemangioma is probably correlated with promoting TSP-1 induced anti-angiogenesis and/ or block nf-kappa B-mediated angiogenesis.