心血管金属材料表面诱导内皮原位修复的微环境构建及动物半体内/体内研究与信号通路探索

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 is now considered the most effective approach to improve the biocompatibility of cardiovascular materials. Due to the key role of materials surface properties contribute to the in vivo mobilization, targeted migration, homing and directed differentiation of pluripotent stem cell, construct a biomimetic microenvironment on materials surface using biomacromolecules and functional factors is now suggested to be a breakthrough in induced endothelium regeneration research. This project intends to integrate chemotactic factors (SDF-1/VEGF) and key proteins (Ln/peptide) in cellular response onto titanium surface to construct a biomimetic microenvironment with the function of induced endothelialization and in-situ regeneration. The property of induced endothelialization will be achieved by controlling the loading quantity and bioactivity of biofunctional molecules. The major signaling pathways between biomacromolecules (factors) and cells during the process of induced endothelialization will be investigated by in vitro and dynamic simulation experiment. 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 evaluate the effect of the microenvironment on endothelium regeneration. Systematic characterization, as well as qualitative and quantitative determination will be carried out by ELISA, PCR,WB，radioimmunoassay, and tissue slice et al to expound the major signaling pathways and molecular mechanisms during the process of microenvironment induced endothelialization and in-situ regeneration. This project will promote the development of cardiovascular materials surface bio-functionalization research.

心血管金属材料被广泛应用, 但其生物相容性远未满足要求。诱导内皮原位修复是改善生物相容性的最有效途径。然而，体内潜能干细胞动员、迁移、靶位归巢及定向分化等都取决于植入材料的表面环境。因此，在表面构建功能微环境是诱导内皮原位修复的突破口。本项目拟将趋化因子（SDF-1/VEGF）和细胞响应关键蛋白（Ln/多肽）引入Ti表面，构建具有内皮诱导与原位修复功能的微环境。通过控制载入量、活性、稳定性等实现原位内皮诱导。借助体外及模拟流场实验探索内皮诱导过程中生物分子（因子）/细胞之间的主要信号通路。通过不同种类（兔和狗）、不同埋置时间（数小时至1年）的动物体内与半体内实验评价微环境诱导内皮及原位修复情况。用酶标、ELISA、PCR、放免、WB等手段进行微环境诱导内皮修复、再生及功能的定性/量检测，阐明微环境诱导内皮及原位修复的主要信号通路与分子机制。本项目将推进心血管材料表面功能微环境研究的进展。

心血管金属材料被广泛应用，但生物相容性远未满足临床要求，比如，支架植入后依然存在晚期血栓和再狭窄。诱导内皮原位修复是改善生物相容性的最有效途径之一，也是近年本领域的研究热点。然而，体内潜能干细胞的动员、迁移、靶位归巢及定向分化等都取决于植入材料的表面环境。因此，在表面构建微环境是诱导内皮原位修复的突破口。本项目将趋化因子和细胞响应关键蛋白引入Ti表面，构建了具有内皮诱导与原位修复功能的四种微环境，包括载SDF-1α肝素/PLL纳米颗粒微环境、载VEGF-肝素/PLL纳米颗粒微环境、基于生物素-亲和素体系的载VEGF-Hep/GREDVY微环境、以及具活性中心功能的SEMA4D/heparin微环境。采用多种检测手段对微环境进行了详细表征。通过控制载入量、活性、稳定性等，对微环境进行了优化。通过多种方法对微环境的血液相容性、细胞相容性及免疫相容性等进行了评价，包括血小板粘附与激活、aPTT及TT测试、纤维蛋白原粘附和变性试验、ECs增殖及趋触、趋化性与迁移试验、巨噬细胞（MA）分泌免疫抑制因子测试等。借助体外及模拟流场实验、动物体内与半体内实验评价了其中两种较优微环境的诱导内皮及原位修复情况。用酶标、ELISA、PCR、放免、WB、组织切片等手段进行微环境诱导内皮修复、再生及功能的定性/量检测，探索了微环境诱导内皮及原位修复的主要信号通路与分子机制。特别地，采用石英晶体微天平（QCM-D）技术研究了动态条件下ECs和MA两种血液循环细胞在微环境表面粘附铺展的动态特征，探讨了不同结构的微环境分子复合物对细胞生理的调控以及多趋化因子对原位内皮化的调节作用。研究表明，优化的微环境能够有效促进原位内皮化，多趋化因子调控下，因子之间具有协同促进作用，但CXCL12对原位内皮化起主导作用。通过项目组的努力，顺利完成了项目研究任务。本项目是基于抗凝-诱导原位内皮化的理念，并模拟机体损伤后的仿生修复原理，在金属材料表面成功构建出功能微环境，取得的进展对材料表面改性具有重要理论价值和实际意义，将推进心血管金属材料表面功能微环境研究的发展。

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