【作者】 王亚宁；

【导师】 万昌秀；

【作者基本信息】 四川大学 ， 生物医学工程， 2003， 硕士

【摘要】 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”和“心包材料表面修饰及内皮化机理研究”两个项目内容的一部分。 心血管对其替代材料提出了较高的要求，材料不但要有适合的物理特性，并且必须具有较好的生物相容性。传统的人造心血管材料在应用中不同程度发生补体激活、凝血等反应，直接或间接地导致血栓的形成，最终导致植入的失败甚至产生并发症危及患者的生命。因此，改善血液相容性是心血管材料研究的重要课题。聚对苯二甲酸乙二醇酯（PET）以其良好的生物化学稳定性和优异的材料力学性能而作为各种生物医学材料得到广泛的应用。不过，由于材料表面的生物相容性问题限制了其更广泛的应用。PET表面不具有—NH₂、—COOH、—OH和其它活性基团，在表面引入其它新分子较困难。如果能在其表面引入RGD，那么其它高分子的接枝就将相对更容易。并且PET是心瓣用涤纶布的原料，选用其作为研究材料具有对血管材料血液相容性研究的代表性。 精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp，RGD）是细胞粘附分子（Cell Adhesion molecule，CAM）中整合素（Integrin）识别和结合配体肽链中的最短序列。将RGD序列结合到材料表面上，来自细胞膜表面的整合素与材料表面四川大学硕士学位论文的RGD通过配体一受体的结合，能在一定程度上促进内皮细胞在材料表曲的粘附、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。 利用EDC作为引发剂成功将光活性叠氮基团引入了RGD短肤，TLC跟踪反应进程并放大分离产物，对Az一RGD合成条件进行了优选，将该产物利用UV接枝于PET膜表面。红外光谱法用以验证合成产物的结构;表面元素分析 （XPS）结果证明膜表面RGD的引入。本论文研究了在PET薄膜上通过丙烯酸光引发接枝RGD，叠氮偶合接枝和Az一RGD光接枝三种方法在材料表面引入具有生物活性的RGD短肤，并对改性后的材料进行了内皮细胞实验以比较其生物相容性。分别采用光学显微镜和电镜观察材料表面的贴壁现象和细胞生长情况，同时采用MTT内皮细胞增殖法证明，PET薄膜表面引入RGD对内皮细胞的生长具有显著的促进作用。模拟血流冲刷实验证明RGD促进内皮细胞在材料上稳定性生长。 结果表明AAc偶合接枝RGD短肤接理论接枝率为34.9%，且有85.20k的一C00H残留在材料表面会对内皮细胞的生长粘附产生负面影响;叠氮苯甲酸的接枝率为35.3%，RGD的偶合接枝率为5.53%，由于偶合反应的产率局限使RGD接枝率不高;Az一RGD偶合接枝RGD的理论接枝率为n .1%，该方法在材料表面不引入残留的多余基团。 接枝AZ一RGD的膜片内皮细胞生长良好，贴壁现象明显，细胞立体结构饱满，细胞伪足伸展且互相连接，贴壁较空白样、AAc偶合接枝样和叠氮偶合接枝样更为牢固。细胞的生长和耐冲刷能力从强到弱的顺序为Az一RGD接枝样>叠氮偶合接枝样>AA。偶合接枝样>AAc接枝样。更多还原

【Abstract】 This article is the part of "Recognizable Molecular Mode on the Polymer Materials Surface Cell and Study on the mechanism of Endothelialization." And it is a Nature Science Fund Project.Polyethylene terephthalate (PET) was widely used in many field, but its bio-compatibility limited it was used in many field, such as biomaterial. Cardiovascular system have higher requirement on biomaterials which be used as substitute. The material must not only have suitable physical capability, but also have good bio-capability. Traditionally, artificial cardiovascular material trend to happen complement activation and blood coagulation when it was exposed to blood. Complement activation and blood coagulation will induce thrombus directly or indirectly. Finally, they result in failure of implant material and the complication will threaten the patient’s life. Therefore it has been an important subject in the materials study to improve the blood compatibility of cardiovascular materials.Arg-Gly-Asp (RGD) is the minimum ammonia serial on the ligand, which can be recognized by most of the integrins, a kind of Cell Adhesion molecule. Connectthe 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 material surface which can promote the blood compatibility of the biomaterials.Three methods were employed to graft RGB on material. The first way was grafting crylic acid on PET film, and intRGDucing RGD on -COOH. Second way was grafting 4-azidobenzoic acid on PET film, and intRGDucing RGD on -COOH. Third way was grafting Az-RGD. After RGD was intRGDuce on surface, endothelial cells were attached to compare their bio-compatibility. 4-azidobenzoic acid and RGD peptide were reacted with EDC, and photoreactive azidophenyl group was intRGDuced on RGD peptide. TLC trail the reaction and separate Az-RGD, and best reactive condition was chosen. UV graft Az-RGD on PET film, and analyze elements of the surface with XPS. And IR analysis also shows RGD was grafted. Microscope and SEM observed endothelial cells on material, and MTT confirmed that RGD could enhance attachment and growth of endothelial cells.Three kinds of material grafted RGD were used to plant endothelial cells. Results show that PET grafted Az-RGD have the best bio-compatibility. It was proved that the material could enhance attachment and growth of endothelial cells. And the material can be cover with endothelial membrane that can enhance its blood compatibility.更多还原