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目的研究玉足海参糖胺聚糖(GAC)抗血栓形成作用的机制。方法分别用不同浓度的 GAG 与人血管内皮细胞株 EA.hy926共孵育24 h;GAG(10 mg/L)或肝素(10 U/mL)分别与 EA.hy926细胞孵育不同时间,检测细胞培养上清中游离组织因子途径抑制物(TFPI)的抗原含量和活性水平;定量测定 EA.hy926细胞内 TFPI mnRNA水平;观察细胞内、细胞表而 TFPI 荧光强度的变化。采用凝血酶、组织型纤溶酶原激活剂(t-PA)、氯化钙建立正常人混合血浆的体外凝块溶解实验,分别加入不同浓度的 GAG 和(或)凝血酶激活的纤溶抑制物(TAFI)特异的抑制剂羧基肽酶抑制物(CPI),观察凝块的凝固和溶解时间,以及 TAFI 相关的凝块溶解延滞(TRR)时间。结果 GAG 以浓度和时间依赖的方式促进内皮细胞 TFPI 的合成、表达和分泌。低浓度 GAG(0.1 mg/L 和 0.5 mg/L)延长了血凝块的溶噼时间,而较高浓度的 GAG(5 mg/L 和10 mg/L)能够缩短血凝块的溶解时间。GAG 以浓度依赖的方式缩短 TRR。结论 GAG 促进血管内皮细胞 TFPI 的合成、表达和分泌,可以抑制 TAFI 功能,促进血栓的溶解。
Objective To study the mechanism of antithrombotic effect of glycosaminoglycans (GAC) from the roots of Yuzu. Methods GAG (10 mg/L) or heparin (10 U/mL) were incubated with EA.hy926 cells for different periods of time, respectively, with different concentrations of GAG incubated with human vascular endothelial cell line EA.hy926 for 24 h. The content and activity level of free tissue factor pathway inhibitor (TFPI) in Qing dynasty; TFPI mnRNA level in EA.hy926 cells was quantified; the intracellular and cell surface changes in TFPI fluorescence intensity were observed. Using thrombin, tissue-type plasminogen activator (t-PA) and calcium chloride to establish a normal human mixed plasma in vitro clot lysis experiment, adding different concentrations of GAG and/or thrombin activated fibrinolytic inhibition TAFI-specific inhibitor carboxypeptidase inhibitor (CPI), observed coagulation and dissolution time of clot, and TAFI-related clot lysis delay (TRR) time. Results GAG promoted the synthesis, expression and secretion of TFPI in endothelial cells in a concentration- and time-dependent manner. Low concentrations of GAG (0.1 mg/L and 0.5 mg/L) prolonged the clot lysis time, while higher concentrations of GAG (5 mg/L and 10 mg/L) reduced the clot lysis time. GAG shortens TRR in a concentration-dependent manner. Conclusion GAG promotes the synthesis, expression and secretion of TFPI in vascular endothelial cells, inhibits TAFI function and promotes thrombolysis.