氧化钛材料与血浆界面反应的分子机制探究

发布时间：2018-11-08 10:55

【摘要】：心脑血管疾病是当今严重威胁人类生命和健康的常见疾病之一。凝血、栓塞与抗凝并发症仍然是临床心血管生物材料就治过程中面临的常见问题,严重制约了此类材料的临床应用。深入研究材料与血液的界面反应过程(特别是材料-蛋白界面作用)；探讨材料介导血栓形成的关键环节和关键因素；从分子学水平揭示材料的抗凝血机理；这些将是新一代具有操控性血液接触类生物材料发展的要求。本课题组在十多年对于钛氧薄膜抗凝血材料的改良应用研究中,积累了丰富的理论和实践研究经验,相应产品已应用临床中。本研究在此基础上,选取具有典型代表意义的高温退火及表面电化学掺杂氧化钛材料,结合先进精密的界面反应手段,原位探讨其材料-血液界面的反应机制,以期实现对其抗凝血机理的深入全面认识。论文主要内容如下：(1)模型氧化钛材料的选取及血液相容性评价：选取两种在实践加工中具有操作简单、对材料表面性质影响较为单一的高温退火及表面电化学n型掺杂处理方法：获得具有不同抗凝血性能的系列氧化钛薄膜及粉末材料；对薄膜和粉末材料的表面物理化学及凝血性能进行了全面表征。结果发现高温退火能导致薄膜表面氧空位的产生,其中尤以800度高温退火最为显著,氧空位的产生(或增加)使得薄膜表面羟基含量增加,碱性羟基的比例同期增加,薄膜的亲水及负电性能增强。对于表面电化学n型掺杂的氧化钛材料也可以通过表面电子的转移,电子的注入能中和材料表面的路易斯酸和质子酸位,使得材料表面的酸性羟基减少,碱性羟基比例增多,材料表面整体的负电性能增加。系列血液相容性评价试验证实800度高温退火及n型掺杂氧化钛材料的血液相容性都较优异。(2)材料血液界面反应的动态原位的监控与探索性研究：利用QCM技术分别研究了表面电化学n型掺杂前后氧化钛材料与血浆各组分(纤维蛋白原、凝血因四十二、贫板血浆及富板血浆)阶段性及全局性的原位作用过程。重点研究了参与凝血行为的重要蛋白质的吸附行为及与血栓形成的关系。发现在热力学驱动下,纤维蛋白原在高浓度下倾向于end-on的可逆吸附行为,而在低浓度下side-on的吸附方式增多。低浓度下,材料与纤维蛋白原的界面作用越明显,在具有更强负电性能的n型掺杂氧化钛表面其吸附方式呈二相吸附,其构象变化中参与凝血的关键位点(激活血小板结合位点、凝血酶作用位点)暴露较少。n型掺杂氧化钛表面更能促进凝血因子十二的激活,能更早的促进凝血的启动,但血栓形成的质量和强度还是主要由纤维蛋白原的吸附状态决定。(3)模型材料与凝血关键蛋白纤维蛋白原的分子作用机制解析：采用精密的界面表征手段(AFM, CD,Microdsc,FCM,免疫化学)结合纳米技术,将模型氧化钛材料与蛋白质的界面反应效应放大,从表面形貌、免疫化学功能、热力学方面解析吸附纤维蛋白原的分子构象。结果显示：在带有更多负电荷及碱性羟基的高温退火及电子转移氧化钛表面,纤维蛋白原更倾向于通过带有正电的αC端形成side-on的方式与材料结合,这种结合可以形成空间位阻,维持D结构域完整,二级结构中α螺旋和β折叠改变较少,避免D结构域中γ链C端血小板结合位点及α链中凝血酶位点的更多暴露,从而降低血栓形成的几率。综上所述。本研究认为提高氧化钛材料的抗凝血性能,可以通过提高其表面的负电性能,调控纤维蛋白原更多的通过其带正电的αC端与材料表面形成side-on的吸附方式,减少血小板结合位点及凝血酶位点的更多的暴露,从而降低血栓形成的几率。本文为钛氧类材料的抗凝血机理的完善提供了数据支持,也为血液接触材料的表面设计提供了新思路。
[Abstract]:Cardiovascular and cerebrovascular diseases are one of the most serious diseases that seriously threaten human life and health. Coagulation, embolization and anti-coagulation complications are still common problems in the treatment of clinical cardiovascular and biological materials, which severely restrict the clinical application of such materials. In-depth study of the interface reaction between materials and blood (especially the role of the material-protein interface), the key and key factors in the formation of material-mediated thrombosis, and the anti-coagulation mechanism of the material from the level of molecular chemistry; These will be a new generation of requirements for the development of blood-contacting biological materials. The research group has accumulated rich theoretical and practical experience in the study of the modified application of the titanium-oxygen thin-film anticoagulant materials for more than a decade, and the corresponding products have been applied in clinical practice. On the basis of this study, the high-temperature annealing and the surface electrochemical-doped titanium oxide material with typical representative meaning are selected, and the reaction mechanism of its material-blood interface is discussed in-situ with the advanced and sophisticated interface reaction means, with a view to realizing an in-depth and comprehensive understanding of its anticoagulant mechanism. The main contents of this paper are as follows: (1) the selection of the model titanium oxide and the evaluation of the compatibility of the blood: two kinds of high-temperature annealing and surface electrochemical n-type doping treatment method with simple operation and single effect on the surface properties of the materials are selected. A series of titanium oxide films and powder materials with different anticoagulant properties were obtained, and the physical and chemical properties and the coagulation properties of the films and the powder materials were comprehensively characterized. The results show that the high-temperature annealing can lead to the generation of oxygen vacancies on the surface of the film, in which the high-temperature annealing of the film is most significant, and the generation (or increase) of the oxygen vacancies causes the hydroxyl content of the surface of the film to increase, the proportion of the basic hydroxyl groups is increased in the same period, and the hydrophilic and electronegative properties of the film are enhanced. The titanium oxide material doped with the surface electrochemical n-type can also be transferred by the surface electrons, the injection energy of the electrons can neutralize the Lewis acid and the proton acid position on the surface of the material, so that the acid hydroxyl group on the surface of the material is reduced, the proportion of the basic hydroxyl groups is increased, and the negative potential of the whole surface of the material is increased. The series of blood compatibility evaluation tests show that the blood compatibility of the 800-degree high-temperature annealing and the n-type doped titanium oxide material is excellent. (2) The dynamic in-situ monitoring and exploratory study of the reaction of the blood interface of the material: The surface electrochemical n-type pre-and post-doping titanium oxide material and the plasma components (fibrinogen and coagulation factor) were studied by using the QCM technique, respectively. The phase and global in-situ action process of the plasma of the lean plate and the plasma rich in the plate. The adsorption behavior of important proteins involved in blood coagulation and its relationship with thrombosis were studied. It was found that in the thermodynamic drive, the fibrinogen tends to be reversible adsorption behavior of end-on at high concentration, while the adsorption of side-on at low concentration is increased. under the low concentration, the more obvious the interface between the material and the fibrinogen, the adsorption mode of the n-type doped titanium oxide surface with the stronger electronegative property is two-phase adsorption, and the conformational change of the n-type doped titanium oxide is involved in the key sites of blood coagulation (activating the platelet binding site, the thrombin action site) is less exposed. The n-type doped titanium oxide surface can promote the activation of the blood coagulation factor 12, and can promote the activation of the blood coagulation earlier, but the quality and the intensity of the thrombus formation are mainly determined by the adsorption state of the fibrinogen. (3) the molecular action mechanism of the model material and the thromboplastin fibrinogen is analyzed, a precise interface characterization method (AFM, CD, Microdsc, FCM, immunochemistry) is adopted to combine the nano technology, the interface reaction effect of the model titanium oxide material and the protein is amplified, and the surface morphology is obtained, the molecular conformation of the adsorbed fibrinogen is analyzed by the immunochemical function and the thermodynamics. The results show that in high temperature annealing with more negative and basic hydroxyl groups and the surface of the electron transfer titanium oxide, the fibrinogen is more likely to be combined with the material through a side-on with the positive C-terminal, which can form a steric hindrance and maintain the integrity of the D domain. in that secondary structure, the change of the spiral and the fold fold is small, so that more exposure of the platelet-binding site in the C-terminal of the C-terminal in the D domain and the thrombin point in the crotch chain can be avoided, so that the probability of the thrombosis is reduced. To sum up. According to the research, the anti-coagulation property of the titanium oxide material can be improved, and more exposure of the platelet binding site and the thrombin site can be reduced by regulating the electronegative property of the surface of the titanium oxide material, regulating the adsorption mode of the fibrinogen more by the side-on with the surface of the material, reducing the binding site of the platelets and the thrombin site, so as to reduce the probability of thrombus formation. The paper provides the data support for the improvement of the anticoagulant mechanism of the titanium-oxygen-based material, and also provides a new way for the surface design of the blood-contacting material.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08

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