表面纤溶系统的机理研究及其在冠脉支架表面的构建

发布时间：2018-11-05 08:16

【摘要】：近年来,血液接触材料在心血管疾病的临床治疗中得到了飞速的发展。但其作为异体材料,植入人体时所引起的血栓问题至今仍未得到妥善解决。大量研究证实,任何一种异体材料在与血液相接触时都会不可避免的引起凝血反应。因此,一些研究者试图通过合理的设计在材料表面模拟人体纤溶系统,赋予材料溶解初级血栓凝块的能力。表面纤溶系统的构建为实现真正意义上的抗血栓材料提供了一种全新的思路。构建表面纤溶系统的关键在于使材料能够从血液中选择性结合纤溶系统核心蛋白质——血纤维蛋白溶酶原(Plg)及其激活剂(如组织型纤溶酶原激活剂(t-PA)),通过二者之间的相互作用产生具有降解纤维蛋白活性的纤维蛋白溶酶,实现对初生血栓的溶解。目前,人们普遍认为ε-氨基和羧基自由的赖氨酸(ε-赖氨酸)对Plg及t-PA具有特异性亲和力,因此,ε-赖氨酸在材料表面的固定将有可能实现表面纤溶系统的构建。基于上述分析,本论文的主要工作是将ε-赖氨酸引入到材料表面,实现表面纤溶系统的构建,并试图从蛋白质水平阐述其作用机理。在此基础上,将ε-赖氨酸化表面的概念应用到冠脉支架上,实现表面纤溶系统在心血管植入材料表面的构建。具体研究内容如下：首先制备了ε-赖氨酸和α-赖氨酸(α-氨基和羧基自由的赖氨酸)修饰的模型金表面,并系统研究了二者与Plg之间的相互作用。采用自组装二肽分子Cys-α-NH2-Lys(CK1)和Cys-ε-NH2-Lys(CK2)的方式实现了ε-赖氨酸和α-赖氨酸在模型金表面的固定。利用水接触角和椭偏厚度测试对改性表面的性质进行表征。通过酶联免疫吸附法(ELISA)比较血浆中的Plg在两种赖氨酸化表面的吸附情况,证实ε-赖氨酸确实能够实现对Plg的选择性结合。通过表面等离子体共振仪(SPR)考察了不同浓度的Plg在两种赖氨酸化表面的动态结合过程。结果表明：ε-赖氨酸化表面对Plg具有更高的结合速率常数和更低的解离速率常数,其亲和常数接近于α-赖氨酸化表面的15倍,从结合动力学的角度进一步证实ε-赖氨酸对Plg具有更高的特异性亲和力。利用纤维蛋白溶酶活性测试,证明该ε-赖氨酸化表面结合的Plg具有更高的纤溶酶转化活性。该研究通过最简单的表面设计,证实了ε-赖氨酸是实现纤溶表面的关键结构,为后续纤溶功能材料的制备提供了理论依据。接下来将对Plg具有特异性亲和力的ε-赖氨酸固定在冠脉支架表面,实现表面纤溶系统在心血管植入材料表面的构建,并对其血液相容性进行了系统研究。采用表面引发原子转移自由基聚合法在L605钻铬合金支架表面聚合甲基丙烯酸2-羟乙酯(HEMA),并将ε-赖氨酸共价接枝在poly(HEMA)内侧链末端。通过X射线光电子能谱测试证实整个改性过程的成功性。同位素标记的纤维蛋白原及白蛋白吸附测试表明poly(HEMA)修饰的L605表而具有极好地排斥非特异性蛋白质吸附的能力,问接法ELISA测试表明poly(HEMA)-Lys修饰的L605表而能够从血浆中选择性结合纤溶酶原。血浆复钙化测试证明该赖氨酸化表面结合的Pig经t-PA激活后能够有效地溶解血浆中的纤维蛋白凝块,而且该纤溶能力具有一定的稳定性和可再生性,即能通过Plg在表面的动态交换保持其活性,进而实现对初生血栓的多次溶解。本研究为解决支架内血栓问题提供了一种的新的思路,同时也为纤溶表面的临床应用奠定了重要的基础。
[Abstract]:In recent years, blood contact materials have developed rapidly in the clinical treatment of cardiovascular diseases. However, as allograft material, the problem of thrombus caused by implantation of human body has not been properly resolved. A large number of studies have shown that any allograft material will inevitably lead to a clotting response when contacted with blood. Therefore, some researchers have attempted to provide the material with the ability to dissolve the primary thrombus block by simulating a human fibrinolysis system on the surface of the material through a reasonable design. The construction of the surface fiber-soluble system provides a brand-new idea for the realization of the anti-thrombotic material in real sense. the key to constructing a surface fiber-soluble system is to enable the material to selectively bind to the fibrinolytic system core protein _ plasminogen (plg) and its activator (e. g., tissue-type plasminogen activator (t-pa)) from the blood, The fibrinolysis enzyme with degradation of fibrin activity is generated by the interaction between the two, and the dissolution of the primary thrombus is realized. At present, it is generally believed that the free lysine (cos-lysine) of glucosamine-amino group and poly-lysine has a specific affinity for Plg and t-PA, and therefore, the immobilization of poly-lysine on the surface of the material will be possible to achieve the construction of a surface fiber-soluble system. Based on the above analysis, the main work of this paper is to introduce poly-lysine into the surface of the material, realize the construction of the surface fiber-soluble system, and try to explain its function from the protein level. Mechanism. On the basis of this, the concept of the surface is applied to the coronary artery stent, which realizes the surface of the surface fiber-soluble system on the surface of the cardiovascular implant material. Construction. Specific research content The model gold surface modified with poly-lysine and poly-lysine was first prepared, and the relationship between them and Plg was studied systematically. The interaction is carried out by the method of self-assembly of the dipeptides of the dipeptide molecules, i.e., CK1 and CK2-NH2-Lys (CK2), in the model gold. Surface fixation. Use of water contact angle and thickness test to test the properties of the modified surface By enzyme-linked immunosorbent assay (ELISA), the adsorption of Plg in plasma on two lysine surfaces was compared. Selective binding. Plg of Plg at different concentrations were examined by surface plasmon resonance (SPR) on two lysine surfaces The results showed that P-lysine surface had a higher binding rate constant and lower dissociation rate constant for Plg, and its affinity constant was 15 times higher than that of the L-lysine surface. The affinity of the opposite sex. Using the fibrinolytic enzyme activity test, it is proved that the Plg of the bound-lysine surface has higher fiber solubility. The enzyme transformation activity was confirmed by the simplest surface design. The key structure of fiber-soluble surface was confirmed by the study, and the preparation of the functional material was provided. A theoretical basis is provided. Next, Plg-lysine immobilized on the surface of the coronary stent is immobilized on the surface of the coronary stent, so that the surface of the vascular implant material is constructed on the surface of the surface fiber-soluble system, and the blood compatibility of the surface fiber-soluble system is compatible with the surface of the coronary stent. A systematic study was carried out. 2-hydroxyethyl methacrylate (HEMA) was polymerized on the surface of L605-drilled chromium alloy stent using surface-initiated atom transfer free radical polymerization. MA) The end of the inside chain. It was confirmed by X-ray photoelectron spectroscopy. The success of the modification process. The isotope labeled fibrinogen and albumin adsorption test showed that HEMA modified L605 table had the ability to repel non-specific protein adsorption, and asked the ELISA test to test the L605 table modified by HEMA-Lys to be selected from plasma. The plasma complex calcification test demonstrated that Pig, which binds to the lysine surface, can effectively dissolve fibrin clots in plasma after t-PA activation, and that the fibrinolysis capacity has certain stability and stability, that is, it can be moved by Plg on the surface. the state exchange maintains its activity, The study provides a new way to solve the problem of thrombosis in the stent, and also provides a clinical application for the surface of the fiber.
【学位授予单位】：武汉理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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