【作者】 赵强；

【导师】 万昌秀；

【作者基本信息】 四川大学 ， 生物医学工程， 2001， 硕士

【摘要】 生物材料在人类的疾病治疗与康复中起着重要的作用。心血管系统的疾病对生物材料的要求较高。传统的人造心血管材料在应用中不同程度发生过凝血、补体激活等反应，直接或间接地导致血栓的形成，最终导致植入物的失败甚至产生并发症危及患者的生命。因此，改善心血管材料的血液相容性，一直是生物材料研究的重要课题。 RGD是大多数细胞表面粘附分子能识别的配体上的最小氨基酸序列，将RGD序列结合到材料表面上，来自细胞膜表面的整合素（Integrin）与材料表面的RGD通过配体一受体的结合，能在一定程度上促进细胞在材料表面的粘附、铺展、生长，最终融合成层。实现在材料表面形成具生物活性的细胞层，提高生物材料血液相容性。 本论文是国家自然科学基金项目：“高分子材料表面细胞可识别分子模型及内皮化机理的研究”的一部分，即在高分子材料表面共价引入人工合成的短肽RGD以达到让内皮细胞与之特异结合并得到更加牢固内皮化表面的目的。 实验进行了甘氨酸、天冬氨酸、精氨酸三种氨基酸的保护；RGD三肽的液相合成与纯化；活性基团-COOH的引入和RGD的接枝；材料的内皮细胞生长实验的研究等。对保护的氨基酸产物用薄层色谱（TLC）进行了反应进程的跟踪及产物纯度的初步鉴定。结合熔点测试、旋光分析及红外等手段对合成产物进行了定性分析。用柱层析的方法对RGD进行纯化，用TLC相对R_f值与美国SIGMA公司标样对照以确认产物。光电子能谱对以羧基为活性基团的接肽反应结果进行分析。光学、电子显微镜观察内皮细胞生长情况以检测接枝短肽的生物活性。 实验得到了叔丁氧羰基（Boc）保护的甘氨酸、苄酯（Bzl）保护的天冬氨酸、硝基（NO₂）保护的精氨酸三种产物并液相合成了RGD三肽，紫外辐照法将活性基团-COOH接到材料的表面，内皮细胞生长实验结果表明，具有生物活性RGD序列已成功接枝到材料表面，并对材料内皮细胞种植起到了促进作用。 建议下一步的工作，应在现阶段初步成功的实验基础上，进行细胞生长情况与RGD或含RGD的更长短肽接枝量的关系，以及在流体力学条件下比较接枝与否与内皮化牢固性、持久性的关系等，从而对内皮化的机理进一步深入研究。更多还原

【Abstract】 Biomaterials play an important role in human disease-treatment and healing. Diseases in cardiovascular system have higher requirement about biomaterials. In the application, traditional artificial cardiovascular materials have blood coagulation, alexin-activation and other effects in different degree which result the formation of thrombus directly or indirectly, and finally lead to the failure of implantation materials or even cause complication and endanger the patients’ lives. Therefore it has been an important subject in the materials study to improve the blood compatability of cardiovascular materials.RGD is the minimum ammonia serial on the ligand, which can be recognized by most of the integrins. Connect the RGD serial on the materials surface, integrin, which comes from membrane, one of the cell adhesion molecule (CAM) on the cell surface, combines with RGD on the materials surface through ligand-acceptor. It can to some extent help the cell adhesion, spreads, grow, and finally mix to layers on the materials surface. Cell layers with biological activities form on the materials surface which can promote the blood compatibility of the biomaterials.This article is the part of "Recognizable Molecular Mode on the Polymer Materials Surface Cell and Study on the mechanism of Endothelialization."which is the Nature Science Fund project. That is covalence induce artificial combine RGD on the polymer materials surface to make endothelial cells to combine with its speciality, so that more fixed endothelializate surface.TLC method was adopted to trace the reaction process and primary test the products purity on the protected ammonia. Analysis over the combined products was carried out with the method of the testing of melting point, polarimeter and IR. RGD was purified with column chromatography. TLC XPS were used to analysis theresults. Optical microscope and SEM observe the status of the endothelial cells growth.Protection over Arg, Gly, Asp were carried and in the experiment Liquid phase synthesis and purification of RGB were taken. Protecting results from Gly protected by Boc, Asp protected by Bzl, Arg by N02 were obtained in the experiment. Pure RGD was obtained in the liquid phase synthesis. Active group -COOH was connected on the materials surface by UV radiation. The endothelialization result shows that RGD serial is successfully connected on the materials surface and improve the effect of materials endothelialization.In the future the study should be focus on the quantitation of cell growth and grafting of RGD, and the grafting of peptides longer than RGD such as RGDX following the way brought forward , and the relation of grafting and endothelialization on the condition of hydrodynamics.更多还原