纳米羟基磷灰石/聚醚醚酮对成骨细胞生物效应的定量蛋白质组学研究

Thermoplastic polymer material polyetheretherketone which has superior physical, chemical and mechanical properties, is a widely anticipated bone implant material. However it usually needs to composite with other materials to improve its bio-inert performance. Hydroxyapatite is regarded as an active material, which is commonly used as composite additives, and the excellent biological properties of nano-hydroxyapatite has drawn more attention. Meanwhile in the field of biomedicine application, greater emphasis is also being placed on the security problem of nanomaterials. The existing medical material safety evaluation methods mostly stays at a cellular level and cannot satisfy the requirements of insight into the interaction between material and the body. The appearance of” –mics” technology make life sciences develop from the single molecule research to a new stage of the high throughput research. It provide an effective way for the study of the integral biological effects and molecular mechanism of the nano-materials and implant materials. By employing quantitative proteomic techniques, this project focus on the comprehensive biocompatibility of nanometer hydroxyapatite composited with polyetheretherketone which contacts directly with osteoblast, to obtain the whole proteins expression regularity and the key signaling pathways, understand the generality and difference of different “bio-inert” substrate materials and to analyze the role of high concentrations of nano-hydroxyapatite mixed with the inert substrate materials in the biological effect from the global molecular level.

热塑性聚合物材料聚醚醚酮具有优越的理化性能，是较受瞩目的骨植入材料，但其需要与羟基磷灰石等生物活性材料复合以改变其生物惰性。研究表明纳米化的羟基磷灰石生物活性更佳，而纳米材料用于人体内的安全性问题备受关注，用其制备的复合材料的最终效应更为重要。现有医用材料标准检测体系中的方法大多停留在细胞水平，不能满足全面深入了解材料与机体相互作用的要求。蛋白质组学技术的发展使生命科学从对单个蛋白孤立研究发展到系统性研究的崭新阶段，为纳米植入材料的全局生物效应评价和分子机理研究提供了有效途径。本课题关注纳米羟基磷灰石与聚醚醚酮复合后对其生物相容性的影响。运用定量蛋白组学技术，研究成骨细胞响应复合材料作用所呈现的蛋白质表达规律、特征蛋白网络及关键信号通路，从分子水平整体层面理解不同“惰性”基体材料生物效应的差异及共性，进一步探讨聚醚醚酮中所含高浓度纳米羟基磷灰石在其生物效应中发挥的作用。

本论文选择主要的骨植入材料类型高分子聚合材料中常用的聚醚醚酮基材料进行研究。采用稳定同位素标记氨基酸培养细胞（SILAC）技术结合质谱鉴定的定量蛋白质组学研究策略，对人成骨细胞MG-63响应各种实验材料的蛋白质整体表达水平进行对比研究，在分子水平整体层面讨论HA作为一种活性添加剂，与高分子基体材料复合后的状态是否能发挥其功能与活性，以及检测游离状态纳米级HA所带来的生物相容性或安全性问题在复合材料中是否存在。对纯PEEK和以PEEK为基底材料共混活性材料羟基磷灰石（HA）制成的复合材料HA-PEEK进行了对比研究。HA-PEEK相对于PEEK的差异蛋白中上调蛋白富集到的功能是钙离子信号相关通路和细胞黏附过程。下调蛋白富集到与mRNA相关的过程，从分子水平解释了HA对PEEK在细胞黏附的促进作用，以及对细胞增殖负性作用。而含钙材料对生物相容性的影响不仅在很小尺寸的游离状态存在，在复合进高聚物后这种效应仍然存在。因而这两类材料产生的生物学效应的差异是由材料表面与成骨细胞直接接触的羟基磷灰石造成的，提示其应用于其他复合材料时的安全性问题依然不可忽视，并且体现出蛋白质组学作为发现纳米材料新的生物效应和分子功能的一项有效工具值得推广应用。