Ti表面共固定肝素和纤连蛋白分子：复合生物功能化的实现

发布时间：2018-05-25 08:57

本文选题：心血管植入生物材料 + 抗凝血　；参考：《西南交通大学》2012年博士论文

【摘要】：心血管植入材料面临的最主要的问题是凝血和血栓发生,因此提高这类生物材料的生物相容性是非常重要和有意义的。目前,通过物理或者化学的方法在材料表面固定抗凝和促内皮细胞粘附的生物分子已经被证实能够较好的抑制血栓发生和促进内皮化,但是多数研究都集中在改善血液相容性或者加速内皮化的某一方面,还很少有同时兼顾抗凝和促内皮化表面构建的研究,而且基本未见有同时兼顾多种生物学功能表面构建的报道。本文选择具有较好生物相容性的Ti作为改性基础,主要采用两种不同的生物化修饰方法在其表面同时固定具有抗凝和促内皮化的生物分子：肝素和纤连蛋白。首先研究了Ti表面肝素和纤连蛋白的层层自组装(LBL),并对自组装膜的生物相容性进行了评价；其次,在此基础上,发展出了单分子自组装肝素和纤连蛋白的方法,借助APTE硅烷化单层以及静电吸引力的作用,在Ti表面构建肝素/纤连蛋白(Hep/Fn)复合生物膜,并对实验条件进行优化。研究了在不同pH (pH7, pH4)以及在有无EDC催化下的Hep/Fn复合生物膜的稳定性和生物功能性,包括血液相容性、内皮细胞相容性、炎症相容性以及抗平滑肌细胞增生性能。最后对所构建的Hep/Fn复合生物膜的生物学功能的机理进行了探讨。综合采用石英晶体微天平(QCM-D)、水接触角分析、傅立叶变换红外光谱(FTIR)、酶联免疫吸附实验(ELISA)、免疫荧光染色分析、扫描电子显微镜(SEM)、原子力显微镜(AFM)、X射线光电子能谱(XPS)等方法对Hep/Fn复合膜的制备过程、生物学活性、成分和性质分别进行了定性和定量表征；通过血小板粘附和激活实验、凝血时间(APTT、PT)实验、纤维蛋白原吸附和变性检测、内皮细胞培养实验、巨噬细胞和细胞因子检测实验以及平滑肌细胞培养实验,全面评价了所构建的复合膜的生物相容性。并通过体外PBS浸泡法以及流动腔实验对制备的复合膜的稳定性以及血小板粘附和内皮细胞附着力进行了初步评价；最后综合借助酶联免疫吸附法、电化学法和等温滴定量热的方法对相关机理问题进行了初步研究。全文主要结果如下： 1.肝素和纤连蛋白可以LBL方式组装在Ti表面且具有一定的稳定性和较好的血液相容性。但是Hep/Fn自组装膜的内皮细胞相容性略差,可能是因为多层膜中肝素的释放所致。LBL构建的多层膜中生物分子释放的控制对于其生物学功能的实现是重要的决定条件。 2.获得了血液相容性和内皮细胞相容性均较好的pH4体系下构建的Hep/Fn复合表面。定量及定性表征显示,尽管肝素和纤连蛋白在不同构建体系(pH7,pH4,EDC/NHS)下样品表面的量无显著差异,但是pH4下肝素结合ATIII的能力最好,纤连蛋白细胞结合位点(RGD)的暴露最多,因此复合物中的生物分子均保持了较好的生物活性,这也是导致后续生物学实验结果差异的主要原因之一。 3.在pH4条件下形成的Hep/Fn复合膜具有较好的稳定性和生物活性。流动状态的下证实此复合生物膜具有较好的血液相容性,且表面粘附的内皮细胞具有较高的稳定性,这为将来的体内应用提供了重要的参考依据。 4.培养内皮细胞和巨噬细胞后的Hep/Fn修饰的样品上的细胞因子表达(TNF-a, MCP-1和IL-1B)要比纯Ti上面的低,共固定Hep/Fn复合物可以减少巨噬细胞的粘附量,从而可以明显的提高材料的抗炎症性能；而且,Hep/Fn复合物可以有效的抑制平滑肌细胞的迁移和增殖,从而抑制植入材料上的内膜增生,但是却不会影响内皮细胞的粘附和生长。研究发现,Hep/Fn复合物促进内皮化和抑制平滑肌细胞增生的机制不同,其机制跟肝素结合到纤连蛋白上导致纤连蛋白构象变化有关。 5.综合利用酶联免疫法、电化学和等温滴定量热法研究了不同pH体系下构建的Hep/Fn表面的肝素和纤连蛋白的生物学活性,并对两种分子的相互作用进行了分析,对样品表面吸附血浆蛋白行为进行了综合分析和比较,同时也研究了肝素和纤连蛋白在溶液中的相互作用过程。获得了对前期不同pH条件下Hep/Fn复合物不同生物学行为较好的解释,对于深入了解不同pH条件下的Hep/Fn表面的不同生物学行为具有十分重要的参考意义和价值。
[Abstract]:The most important problems faced by cardiovascular implant materials are coagulation and thrombosis, so improving the biocompatibility of such biomaterials is very important and meaningful. At present, biomaterials with physical or chemical methods to immobilization anticoagulant and promote endothelial cell adhesion on the surface of the material have been proved to be able to inhibit the blood. Thrombus occurs and promotes endothelialization, but most studies focus on some aspects of improving blood compatibility or accelerating endothelialization. There are few studies that have both anticoagulant and endothelialization surface construction, and there have been no reports of various biological functional surface construction at the same time.
In this paper, we chose Ti with good biocompatibility as a modification basis, mainly using two different biological modification methods to immobilization of anticoagulant and endothelialized biomolecules on the surface of them: heparin and fibronectin. First, the layer self assembly (LBL) of heparin and fibronectin on the surface of Ti was studied, and the biology of self assembled monolayers was studied. The compatibility was evaluated. Secondly, a single molecule self-assembled heparin and fibronectin were developed on this basis. With the help of APTE silane monolayer and electrostatic attraction, heparin / fibronectin (Hep/Fn) composite biofilm was constructed on the Ti surface, and the experimental conditions were optimized. The study on different pH (pH7, pH4) and at the same time was carried out. The stability and bioactivity of the Hep/Fn composite biofilms with or without EDC catalysis, including blood compatibility, endothelial cell compatibility, inflammatory compatibility and anti smooth muscle cell proliferation. Finally, the mechanism of the biological function of the constructed Hep/Fn composite biofilm was discussed. Quartz crystal microbalance (QCM-D), water was used. Contact angle analysis, Fu Liye transform infrared spectroscopy (FTIR), enzyme linked immunosorbent assay (ELISA), immunofluorescence staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and X ray photoelectron spectroscopy (XPS) were used to characterize and quantitatively characterize the biological activity, composition and properties of the Hep/Fn composite membranes, respectively. Through platelet adhesion and activation experiments, coagulation time (APTT, PT) experiments, fibrinogen adsorption and denaturation detection, endothelial cell culture experiment, macrophage and cytokine detection experiments, and smooth muscle cell culture experiments, the biocompatibility of the composite membrane was evaluated comprehensively. In addition, the PBS immersion method and the flow chamber were used in vitro. The stability of the composite membranes and the platelet adhesion and adhesion of endothelial cells were preliminarily evaluated by the experiment. Finally, the related mechanism problems were preliminarily studied with the help of enzyme linked immunosorbent assay, electrochemical method and isothermal titration calorimetry. The main results are as follows:
1. heparin and fibronectin can be assembled on the surface of Ti with LBL and have a certain stability and good blood compatibility. But the compatibility of the endothelial cells of the Hep/Fn self assembled membrane is slightly poor. It may be because the release of biomolecules in the multilayer membrane constructed by the release of heparin in the multilayer membrane is the result of the realization of its biological function. It is an important determinant.
2. the Hep/Fn composite surface constructed under the pH4 system with good blood compatibility and endothelial cell compatibility was obtained. Quantitative and qualitative characterization showed that although heparin and fibronectin had no significant difference on the surface of the samples under different construction systems (pH7, pH4, EDC/NHS), the ability of heparin to bind ATIII under pH4 was the best, and fibronectin cell junction was the best. The RGD is exposed most, so the biomolecules in the complex maintain good biological activity, which is one of the main reasons for the differences in the results of subsequent biological experiments.
3. the Hep/Fn composite membrane formed under the condition of pH4 has good stability and biological activity. The flow state confirms that the composite biofilm has good blood compatibility and the surface adhered endothelial cells have high stability. This provides an important reference for future application in vivo.
4. the expression of cytokine expression (TNF-a, MCP-1 and IL-1B) on Hep/Fn modified samples after cultured endothelial cells and macrophages is lower than that on pure Ti. Co immobilization of Hep/Fn complex can reduce the adhesion of macrophages, which can obviously improve the anti-inflammatory properties of the material; moreover, the Hep/Fn complex can effectively inhibit the smooth muscle of the smooth muscle. The migration and proliferation of cells inhibit intimal hyperplasia on the implanted material, but it does not affect the adhesion and growth of endothelial cells. It is found that the mechanism of the Hep/Fn complex promotes endothelialization and inhibits the proliferation of smooth muscle cells. The mechanism is related to the conformation of fibronectin to the fibronectin, which is combined with heparin.
5. the biological activity of heparin and fibronectin on the surface of Hep/Fn under different pH systems was studied by enzyme linked immunosorbent assay, electrochemistry and isothermal titration calorimetry, and the interaction between the two molecules was analyzed. The adsorption of plasma protein on the surface of the sample was analyzed and compared, and the heparin and the heparin were also studied. The interaction process of fibronectin in the solution has obtained a good explanation for different biological behavior of Hep/Fn complexes under different pH conditions. It is of great reference significance and value for understanding the different biological behavior of Hep/Fn surface under different pH conditions.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

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