纳米表面壳聚糖-胶原复合膜对肝细胞P450酶诱导性的调节及其机制

P450 induction based drug-drug interactions are often associated with drug toxicity, treatment failure and drug withdrawal. Primary hepatocyte model, which uses collogen as substratum, is regarded as "gold standard", and widely used to assay inductive potential of drugs. However, it is a completed, money consuming model with big inter-individual deviation. National biodegradable chitosan has been successfully applied in liver engineering, thanks to its favorable features. But no systemic study has been reported about their effects on inducibility of hepatocyte P450. Our study has indicated that physically modified nano-chitosan features an increased ability to promote cellular adhesion, growth, proliferation and differentiation. Considering the shortcomings of the above mentioned hepatocyte induction-assaying model, and favorable properties of modified chitosan, this project is to prepare chitosan-collogen substratums with nano-convexity, and systemically study their modulatory effects of on inducibility of hepatocyte P450 in present typical inducers. The underlying signal pathways in which nuclear factors participate are also interesting targets. The output of this project includes protocols for preparation of nano-convex chitosan-collogen substratums which can optimize inducibility of hepatocyte P450 and an optimized DDI evaluation model, which is both theoretically and practically important.

临床用药引起的P450诱导常导致药物毒性、治疗失败和药物撤市等严重后果；原代肝细胞是评价药物诱导性相互作用的 "金标准"，但该模型中细胞培养过程复杂、成本昂贵、个体变异大。天然可降解材料壳聚糖或改性壳聚糖在肝脏组织工程中已取得良好的应用效果，但尚未见关于其调节肝细胞P450诱导性的系统研究报告。申请人已有研究表明纳米表面改性壳聚糖膜材能进一步促进细胞的黏附、生长、增殖、分化。本项目拟在已有工作基础上，探索和优化纳米表面壳聚糖-胶原复合膜的制备工艺，研究该膜对肝细胞P450诱导性的调节机制，阐明其中的核受体信号通路。研究结果预期获得对肝细胞P450诱导性具有优化调节作用的纳米表面壳聚糖-胶原复合膜制备工艺，并依托新工艺和新材料建立创新的肝细胞P450诱导评价模型，具有重要的理论意义和实践价值。

壳聚糖是具有良好生物功能的可生物降解高分子材料，经物理和化学改性可获得一些新的生物学功能。原代肝细胞是评价药物诱导潜性的通量化模型，目前经典的模型以胶原铺板进行细胞培养。本项目建立了小鼠原代肝细胞分离培养技术，细胞分离活率达到85%，基于肝细胞模型建立了384孔板亚通量CYP诱导性相互作用检测平台；评价了不同脱乙酰度、不同共混比例、不同铺制方法的壳聚糖/胶原复合膜材对肝细胞的生物相容性；引进和建立了PXR与PPARa敲除小鼠模型用于CYP诱导及代谢性疾病模型；基于建立的肝细胞和PXR小鼠等模型开展了CBZ、纳米材料等对CYP及肝脏代谢的调控作用。. 本项目开展过程中，主持人共发表第一作者和通讯作者SCI论文3篇，通讯作者中文核心期刊论文1篇，合作方第一或通讯作者发表SCI论文2篇及中文核心期刊论文4篇，获得3项专利授权，联合培养硕士研究生3人(在读)，主持人论文2篇次获得宁波市自然科学优秀论文三等奖和优秀奖，超额完成了既定的目标。. 本项目在考察壳聚糖/胶原复合膜材对肝细胞诱导模型的影响时发现，壳聚糖的脱乙酰度对肝细胞的粘附和生长具有重要的影响，脱乙酰度越高，细胞的粘附生长状态越好，但即使脱乙酰度达到90%，其对细胞仍有一定毒性，细胞培养24h后大量脱壁，说明壳聚糖用于培养原代肝脏细胞还有待通过物理化学手段进一步改善其生物相容性。基于本项目引进和建立的PXR敲除小鼠模型和通量化QPCR检测平台，可以快速大量的进行药物对CYP的诱导潜性评价，并发现他们与PXR间的关系，从而具有用于商业化技术服务的应用前景。另外，纳米材料在生活中的应用日益广泛，如纳米TiO2在大量的医用品和生活用品中都有应用，其经典的安全性评价方法包括常见肝功、肾功和组织病理学等手段，本项目通过小鼠实验证实胆红素水平及肝脏CYP和转运体的表达水平可能是评价纳米材料的敏感指标，值得进一步验证和应用。