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目的利用单分子力谱法检测香烟提取物(cigarette smoke extract,CSE)对血栓调节蛋白(thrombomodulin,TM)与凝血酶间单分子水平相互作用,探讨吸烟致血管内血栓形成机制。方法构建TM-GFP质粒并转染至COS-7细胞,应用原子力显微镜(atomic force microscope,AFM)分组测力:(1)空白质粒对照组(GFP-Thr);(2)TM-Thr对照组;(3)5%CSE孵育TM-Thr细胞组(CSE-TM-Thr);(4)5%CSE孵育空白质粒细胞组(CSE-GFP-Thr)。结果 (1)TM与凝血酶的相互作用力为(60.90±0.82)pN,成键几率为(22.58±3.95)%,抗体阻断后成键几率为(2.58±2.0)%。(2)与TM-Thr组比较,GFP-Thr组、CSE-TM-Thr组、CSE-GFP-Thr组,3组AFM成键几率明显减低,P<0.05;但3组间两两比较差异无统计学意义。在定量TM修饰的硅片表面检测证实CSE同样可减少TM与凝血酶的成键几率。结论吸烟可能通过减少TM与凝血酶结合几率,抑制TM的抗凝作用,从而导致血管内血栓形成。
Objective To detect the interaction of cigarette smoke extract (CSE) with monomolecular level between thrombomodulin (TM) and thrombin by single molecule force spectrum and investigate the mechanism of thrombosis induced by smoking. Methods TM-GFP plasmid was constructed and transfected into COS-7 cells. The force was measured by atomic force microscope (AFM): (1) blank control group (GFP-Thr); (2) TM-Thr control group ; (3) CSE-TM-Thr cells were incubated with 5% CSE; (4) CSE-GFP-Thr cells were incubated with 5% CSE. Results (1) The interaction between TM and thrombin was (60.90 ± 0.82) pN, the probability of formation of bond was (22.58 ± 3.95)%, and the probability of formation of antibody after blocking was (2.58 ± 2.0)%. (2) Compared with TM-Thr group, the bonding probability of AFM in GFP-Thr group, CSE-TM-Thr group and CSE-GFP-Thr group was significantly decreased No statistical significance. Quantitative TM modified silicon surface test confirmed that CSE can also reduce the TM and thrombin bond formation probability. Conclusion Smoking may reduce the anticoagulant effect of TM by reducing the chance of TM binding to thrombin, leading to intravascular thrombosis.