基于OATs转运蛋白和DNA嘌呤加合物的中药肾毒性分析模型的建立与评价

The renal toxicity of traditional Chinese medicines (TCM) has become the one of most important problems for the safety of TCM, which can induce the progressive renal interstitial fibrosis and urothelial malignancies. The traditional analysis of nephrotoxicity components and the safety evaluation of TCM are relatively independent, and both of them own many disadvantages, such as heavy workload, time-consuming, low efficiency and high cost. Based on the previous work, we intend to establish a simple, rapid and efficient model for the analysis of toxic components in TCM and the evaluation of TCM safety, which will imitate the interaction of drug and target in vivo. In this model, the multidimensional liquid chromatography/mass spectrometry, molecularly imprinted technology and fluoroimmunoassay will be integrated together. This model consists of two parts. 1. A target recognition unit will be established for the primary screening of the potential nephrotoxicity component from TCM. In this unit, the two dimensional liquid chromatography/mass spectrometry will be used as the supporter, and the transmembrane transporter OATs will be used as the identifying medium. 2. A supersensitive fluorescence immunoassay unit will be established to screen the potential nephrotoxicity component from TCM again, in which the purines of DNA are the identifying medium. The model will be evaluated by positive drug and assessed through the in vitro cytotoxicity test and in vivo animal assay. This model will realize the rapid screening of the toxic components in TCM, which can induce the renal interstitial fibrosis and urothelial malignancies. This model combines recognition, separation and analysis into one model, and it will provide a new idea and method for the analysis and evaluation of the safety of TCM.

肾纤维化和泌尿系统癌症等肾毒性反应已成为中药安全性主要问题之一，然而传统的中药肾毒性成分分析与评价方法相对独立，且均存在工作量大、耗时长、评价效率低和成本高等问题。本研究拟在前期工作基础上，整合多维液相色谱-质谱联用、分子印迹和荧光免疫分析技术，仿生模拟体内药物与靶点相互作用，建立一种简便、快速、准确的集毒性成分分析和评价于一体的中药肾毒性分析模型。该模型包含①以已知的肾毒性成分跨膜转运体OATs为识别介质，建立二维液相色谱/质谱联用靶向识别单元，对具潜在致肾纤维化和癌症的中药毒性成分进行一次筛选；②以DNA嘌呤碱基为识别介质，制备荧光超敏免疫分析单元，对目标物进行二次筛选。采用阳性药物验证实验、体外细胞实验和整体动物实验对模型进行评价。拟建立的模型集毒性成分“识别-分离-分析”于一体，实现对中药中致肾纤维化和致癌毒性成分的快速筛查，可为中药肾毒性成分的分析和安全性评价提供新的思路和方法。

研究将分子印迹技术、材料表面修饰技术、固相萃取技术和二维液相色谱技术相结合，采用表面印迹法、戊二醛交联法、EDC/DMAP反应，以甲基丙烯酸、碱基嘌呤等为功能基团，制备了对肾毒性物质马兜铃酸具有特异性识别能力、稳定性高、重复性好的分子印迹聚合物（MIP）及功能化材料；分别采用扫描电镜、透射电镜、红外光谱、元素分析、热重分析、氮吸附-脱附、X-射线光电子能谱、X-射线衍射等技术，测定了所制备的MIP和功能化材料的表观形貌、物理化学性质和热稳定性；结合高效液相色谱法、紫外分光光度法、荧光分光光度法、液质联用法或凝胶电泳法等测定了MIP和功能化材料对目标物质的特异识别能力，优化了识别条件，并考察了分子识别的机制和基本规律。以所制备的MIP和功能化材料为识别介质，与固相萃取技术和二维色谱技术相结合，建立了中药中肾毒性物质的筛选模型，成功应用于中药中可疑性肾毒性物质的筛选，为中药中肾毒性物质的筛选、分离和分析提供了新的方法和思路。