用PAMAM介导心血管材料表面于(半)体内在体形成三维微环境及促进血管内膜原位自修复的分子机制

Cardiovascular metal biomaterials have been extensively used in clinical treatment of cardiovascular diseases. However, the biocompatibility of devices is remaining far from perfect. Induced in-situ endothelium regeneration based on the surface microenvironment construction is now considered the most effective approach to improve the biocompatibility of cardiovascular materials. The current microenvironment research mainly relates to biomimetic construction under the conditions of in vitro, static and two dimensions, and the formed microenvironment is different to the actual biology microenvironments outside the cells. So the three dimensions microenvironment with perfect functionallty is becoming a research hotpot. Based on our research experience and the features of the physiology environments with the vessel and blood, this project will screen out and modify the dendritic polyamidoamine (PAMAM) onto the titanium or stainless steel surface. Then, it focuses on the deformation of biological microenvironment mediated by dendritic macromolecule on different biological states. The in vitro dynamic simulation experiment related to the fluids such as plasma, blood etc will be performed. In vivo and ex vivo study will be performed in various animal models (rabbit and dog) with different embedding time (hours to one year) to investigate the details of the microenvironment formation. Systematic characterization, as well as qualitative and quantitative determination will be carried out by ELISA, PCR,WB，radioimmunoassay, and tissue slice et al. According to the expounded major characteristics of three dimensions microenvironment, the molecular mechanisms during the formation of microenvironment and the in-situ endothelialization and regeneration on cardiovascular metal biomaterials surface will be disclosed. This project will promote the development of cardiovascular materials surface bio-functionalization research.

金属植入器件在心血管疾病治疗中被普遍应用，但生物相容性亟待提高。表面微环境构建是改善其生物相容性的有效途径。已有研究主要为“体外、静态、二维”仿生，其形态和功能与实际细胞外微环境有较大差异。构建具体内特征、有组织与环境响应功能的三维微环境，是本领域重要方向。结合血管/血液界面特点，基于已有积累，申请者在心血管金属材料表面引入树枝状大分子聚酰胺胺PAMAM，进行分子端部设计与调控后，利用(半)体内环境，介导材料表面在体、原位形成细胞外三维微环境。项目选择体外动态血浆/全血模拟状态、动物半体内血流状态、动物体内血液循环状态等三种不同生物学环境，借助酶标、ELISA、PCR、放免、WB等手段，研究PAMAM-X介导材料表面在实际环境下“在体形成”细胞外“三维”微环境的动态过程、蛋白分子行为以及对血管内膜原位自修复的影响，揭示微环境在体形成机制。本项目将推进心血管生物材料表面功能化的研究与应用。

针对金属植/介入器件生物相容性亟待提高的临床需求，为了在表面形成更接近细胞外实际特征的微环境，项目探索了体内、动态、原位在体形成三维微环境的可能性，取得了重要结果。（1）项目从动力学与热力学角度，研究并获得了三种端基PAMAM-X分子与主要血浆蛋白间的反应特征与热力学参数，通过定性定量对比，明确了第三代（G3）、端基为-NH2/-COOH 的PAMAM树枝分子是介导分子首选，当介导血浆蛋白与其复合时，更利于蛋白构象维持；明确了氨基密度为1.0 mg/ml时表面抗血栓性好，Ac-SDKP功能化PAMAM-NH2后，表面内皮细胞活性增加。（2）体外动态模拟研究证实，一元蛋白环境中PAMAM-NH2表面吸附更多BSA与γ-球蛋白，二元蛋白环境中BSA或γ-球蛋白会影响纤维蛋白原在PAMAM-COOH表面的吸附，三元蛋白环境下PAMAM-NH2表面三元混合蛋白吸附量与介导分子密度正相关，但PAMAM-COOH表面介导分子密度与蛋白吸附量负相关；确定了表面有效调控蛋白行为、且生物相容性好的最优PAMAM-NH2/COOH引入量。（3）动物半体内、体内研究证实， N1.0样品表面血液相容性优异，低密度Ac-SDKP引入不影响血液相容性，但高密度引入将导致血栓形成；样品植入腹主动脉30天后，Ac-SDKP-PAMAM-NH2表面炎性反应弱，抑制增生，新生内膜薄而光滑，表达连续CD31细胞层，内皮化完整近似正常血管内壁。通过分析样品表面各血浆蛋白吸附特征、择优吸附行为等，揭示了PAMAM-X介导蛋白参与微环境形成的本质，初步认识了表面在体形成微环境及诱导内膜原位修复的机制。项目发表论文16篇（SCI 11篇），获授权专利10项，获中国生物材料学会科学技术奖一等奖1项。项目研究进展与成果，为植/介入器械表面介导设计、在体微环境创建提供了依据和新途径，推进了生物材料表面功能化研究与发展。