摘要
目的观察体外在血栓制作中应用凝血酶对血栓溶解性的影响及其机制;对血浆纤维蛋白原水平与血栓中交联纤维蛋白含量进行相关分析,预测血栓溶解性的价值。另外,提出一种精确测定血栓中交联纤维蛋白含量的方法。方法 (1)红细胞比容35%～45%的健康人新鲜静脉血10 ml,平分为5份,取其中4份,3份分别加入高(75 u/ml)、中(50 u/ml)、低浓度(25 u/ml)凝血酶,1份加入生理盐水作为对照,摇匀,置于37℃水浴箱中3 h,使其凝固形成血栓。(2)剩余的1份血标本,应用Clauss法检测人血纤维蛋白原水平。(3)观察4组血栓之间机械性能的差别,包括弹性和塑性。(4)每个血栓分别切取一部分,修剪成重量为150 mg的正方体,磷酸缓冲液冲洗后,置于纤溶酶溶液中,在37℃水浴箱中孵育振荡,肉眼观察血栓降解过程。(5)记录血栓降解时间(包括血栓块消失时间和交联纤维蛋白絮状物消失时间),离心,取上清液,保存于-20℃。(6)解冻上清液,用蒸馏水稀释,应用酶联免疫法检测D二聚体浓度。(7)根据D二聚体浓度计算血栓中交联纤维蛋白含量。(8)对4组血栓交联纤维蛋白含量进行方差分析并两两比较;对4组血栓降解时间进行秩和检验并两两比较;对血浆纤维蛋白原水平与高凝血酶组血栓中的交联纤维蛋白含量进行相关分析。结果无凝血酶组血栓韧性差,易碎;应用凝血酶的3组血栓均具有良好的韧性。与无凝血酶组相比,高、中、低浓度凝血酶组血栓的降解时间和交联纤维蛋白含量均显著增加(P均<0.01);高、中浓度凝血酶组与低浓度凝血酶组相比,血栓的降解时间和交联纤维蛋白含量均显著增加(P均<0.01);高浓度凝血酶组与中浓度凝血酶组之间相比,血栓降解时间和纤维蛋白含量差异无统计学意义(P均>0.05)。血浆纤维蛋白原水平与高浓度凝血酶组血栓的交联纤维蛋白含量呈直线相关(r=0.681,P<0.001)。结论在体外
Objective To observe the effect of thrombin concentrations on the lysability of the thrombus,and clarify whether the underline mechanism involves the change in cross-linked fibrin content of the thrombus;the relationship between the level of plasma fibrinogen and the content of cross-linked fibrin of the thrombus was analyzed to determine the predictive value of the plasma fibrinogen level for the lysability of the thrombus.Intriguingly,that allows to precisely determine the content of cross-linked fibrin in thrombus. Methods(1)Venous blood of 10 ml was drawn from healthy volunteers with erythrocrits 35%～ 45%,and the sample was divided equally with syringes into five portions.,three were treated with low(25 u/ml),medium(50 u/ml)and high(75 u/ml)concentration of thrombin-containing solutions,respectively,and one portion was treated with saline.After vortex,the reaction systems were placed in water bathes at 37℃ for 3 h during which the thrombi were formed by coagulation process.(2)The plasma fibrinogen level was measured with Clauss method using the remained one portion of the blood sample.(3)After removing thrombi from syringes,the mechanical properties of the thrombi,including elasticity and plasticity,were compared.(4)A portion of each thrombus was cut and trimmed into cubes weighting around 150 mg.After washing with phosphoric acid buffer solution,the cubes were incubated in plasmin-containing solutions at 37℃ with gentle rocking,and the degrading processes were observed with naked eyes.(5)The degradation times of thrombi,including the disappearing times for both mass and fibrin floss of thrombi,were recorded.After centrifuging,the supernatant was collected and stored at-20℃.(6)After the iced supernatants were thawed and diluted,D-dimmer concentrations were measured with ELISA.(7)The cross-linked fibrin content of thrombi was calculated from D-dimmer concentrations in reaction systems,a method that is proposed for the first time to our knowledge.(8)Variance analysis of fibrin content,rank test of degra
出处
《血栓与止血学》
2010年第6期252-258,共7页
Chinese Journal of Thrombosis and Hemostasis
