基于体内生物屏障细胞模型-微流控芯片的中药与转运体相互作用的分析新方法研究

Potential drug-drug interaction should not be ignored in combination use of TCM and Western medicine. Drug transporters play an important role in drug-drug interaction. There exist many active drug transporters on the biological barriers in vivo. However, few methods can predict the interaction between TCM and drug transporters effectively and rapidly. This project aims to develop the microfluidic chip system with biological barrier cell models, mainly focused on intestinal absorption barrier (Caco-2 cells and MDCK-MDR1 cells) and blood-brain barrier (cerebral microvascular endothelial cells). The cellular efflux transporters are characterized. The static and dynamic cell culture, trans-membrane electrical resistance measurement and online fluorescent detection are integrated in the device, which would be used for rapid and high-throughput assay of interaction between Chinese medicine active ingredients and drug transporters. Moreover, microscale cell culture followed by the multiple gradient generator and on-chip solid-phase extraction columns for sample pretreatment prior to mass analysis are integrated in the device. The quantitative characterization of the interaction between Chinese medicine ingredients and transporters are to be obtained using an integrated microfluidic device coupled to a mass spectrometer. Therefore, the potential substrates, inhibitors or inducers would be clarified, and the potential effects of related transporters on the combination of TCM and Western medicine be evaluated. The aim of this project is to explore innovative methods based on microfluidic chips for the rapid analysis of the interaction between Chinese medicine ingredients and drug transporters, which would be better than the in vitro cell models. This project will provide technical references and scientific evidences for clinical combination of TCM and Western medicine and guarantee clinical medication reasonable and safe.

中西药联用潜在的药物-药物相互作用不容忽视，药物转运体是引发这种相互作用的重要因素。体内生物屏障上的药物转运体非常活跃。但是中药成分与药物转运体的相互作用预测尚缺乏快速有效的手段。本课题拟构建基于微流控芯片的体内生物屏障细胞模型，主要有肠道吸收屏障（Caco-2细胞/MDCK-MDR1细胞）和血脑屏障模型（脑微血管内皮细胞），表征其外排型转运体，集成细胞静态和动态培养、跨膜电阻测定和在线荧光检测等功能，实现快速、高通量的中药成分与转运体相互作用分析；进而结合液质联用技术，集成细胞三维培养、药物浓度梯度生成、样品前处理等功能，对中药成分与转运体相互作用进行定量表征，阐明其可能的底物或者抑制剂、诱导剂，评价转运体在中西药联用时的潜在效应。本项目旨在探索中药成分与转运体相互作用的快速分析的创新方法，建立优于体外细胞模型法的微流控芯片系统，为临床上中西药联用的合理安全提供技术借鉴和科学依据。

本项目围绕血脑屏障和肠道吸收等部位的药物转运体，以P-gp为代表，借助于微流控芯片技术的支撑，结合不同的细胞模型，建立中药成分与药物转运体的相互作用预测新方法，主要研究内容和结果有：（1）成功构建了以脑微血管内皮细胞为核心、高保真的血脑屏障（BBB）微流控芯片模型，包括单细胞培养BBB芯片模型和多细胞共培养BBB芯片模型，实现了三种细胞动态培养和流体剪切应力模拟，进行了BBB屏障功能、转运蛋白功能等表征；（2）基于以上两种BBB芯片模型进行了多个中药活性成分跨BBB转运特征研究，及其与转运体P-gp相互作用，结果表明芯片模型更接近体内真实生理状态，具有更好的应用潜力；（3）成功构建了体外Caco-2细胞模型和MDCK-MDR1细胞模型，表征P-gp，对中药知母进行了双向转运研究，获得肠道吸收转运的特征信息，探明与转运蛋白的相互作用；（4）基于液滴微流控，在水相引入浓度梯度生成器，建立了浓度梯度液滴微流控芯片平台，可应用于抗白念珠菌药物等筛选研究；（5）成功建立了慢病毒载体膜蛋白稳定策略结合SPR的中药成分与P-gp相互作用研究方法，以慢病毒作为P-gp的载体，可保持其天然构象，方法专属性强，并可缩短筛选时间；（6）首次成功构建了整合血脑屏障BBB和脑胶质瘤U251细胞的BBB-U251-芯片模型，实现了中药中抗脑胶质瘤潜在成分跨血脑屏障转运和抗脑胶质瘤药效的同时评价，为抗脑胶质瘤潜在药物筛选提供了更加接近体内生理环境、切实可行的模型。本项目建立了优于体外细胞模型法的体内屏障细胞模型-微流控芯片系统，可为临床上中西药联用的合理安全提供科学依据和技术借鉴，并有望应用于中枢神经系统新药研发。已发表学术论文20篇，其中SCI论文9篇；项目负责人应邀在国内学术大会作主题报告6次；培养了9名研究生；申请国家发明专利4项；获全国和上海市优秀论文奖共5项。