基于特异性敲除技术的芍药甘草汤配伍机制的研究

It is an essential issue that we need to answer what the relationship between the interactive function of the chemical components in prescriptions with the compatibility theory of prescription is like. Through the years of research on compatibility theory of classical prescription, based on the small molecule monoclonal antibody technology platform, we have developed paeoniflorin and glycyrrhizin specific knockout technology using monoclonal antibody against paeoniflorin and glycyrrhizin to interpret the compatibility theory underline the classic prescription. On basis of animal and cellular models corresponding with Shaoyaogancao Decoction traditional attending efficacy, in vivo and in vitro, static and dynamic, pharmacodynamic and material combined evaluation criteria, through the differences analysis in the comparative study on plasma concentration and target organ specific distribution characteristics changes and in vivo, the relationship of dose, effect, route, location, as well as the overall analgesic antispasmodic effect of differences in vitro relationship of complete ingredients of Shaoyaogancao Decoction, and paeoniflorin, glycyrrhizinknockout extracts, we can clarify the regulation of compatibility among different components and interpret the contributions of paeoniflorin and glycyrrhizinin in Shaoyaogancao Decoction to the compatibility, as well as the pharmacodynamics and distribution characteristic in target nuclei. The realization of these goals will further the development of technology route and research ideas for small molecular monoclonal antibody technology used in Chinese medicine prescription compatibility theory and the material basis of research areas. Further more ,we can provide a successful example of the expansion of the application of this new technology in the field of Chinese medicine research.

化学成分间的互动作用关系是方剂配伍机制研究需要回答的基本问题,特异性敲除技术可对方剂整体中所含中药成分间的互动关系进行研究。本课题以经方配伍研究的多年积累为基础，以自主建立的中药小分子单抗技术平台为保障，以经典名方芍药甘草汤为模板：采用小分子单抗特异性敲除技术，利用已获得的芍药苷和甘草酸单抗，建立特异性敲除芍药苷和甘草酸的技术方法以及免疫分析方法；为今后借助与芍药甘草汤传统主治功效相对应的动物和细胞模型，通过比较定量敲除芍药苷、甘草酸前后，在体内血药浓度及脑内亚器官特异性分布特征上的变化，以及整体、离体镇痛解痉效应的差异，揭示二者在量-时-位-效方面的相互关系，进一步阐明芍药甘草汤的配伍机制奠定基础。

本研究利用自主建立的中药小分子单克隆抗体技术，以经典名方芍药甘草汤为模板，选取其代表性指标成分芍药苷、甘草酸，人工合成免疫原，通过免疫动物，阳性克隆筛选，细胞融合等分别建立了抗芍药苷和甘草酸的单克隆杂交瘤细胞株，并通过大量制备特异性抗体，分别建立了相应的免疫分析方法。该方法可以分析芍药甘草汤中单一有效成分的体内过程，也可以实现多种成分在单器官或组织中，或在多器官或组织样品的含量或存在的同步检测。不仅能够通过解析芍药甘草汤主成分的体内过程及其相互作用关系，进一步阐释其配伍机理的科学内涵，还解决了取材量较少的生物样品多成分分析检测的技术难点。此外，本研究还分别制备了芍药苷、甘草酸的免疫亲和色谱柱，并成功敲除了含芍药苷和甘草酸的提取液，为进一步完成芍药甘草汤的药效物质基础研究奠定了坚实的基础。