基于热稳态调控网络节点-TRP通道辨识中药四性的生物学基础

The theory of natures of Chinese herbs is of paramount importance and its biological significance is tough to explore. Our previous experimental results demonstrated that some correlation between the natures of Chinese herbs and the thermo transient receptor potential (thermo-TRP) channels must exist over the process of carrying out our subject supported by the national natural science foundation of China. To further our study, a set of in vitro system for detecting and analysing both the relationship and the characteristics thereof between thermo-TRP channels and natures of Chinese herbs need to be established urgently. Thus, a plan to construct a novel system containing both dorsal root ganglion (DRG) neurons expressing endogenous thermo-TRP channels and HEK 293 cells expressing exogenous thermo-TRP channels is put forward in this subject with a focus on the the subtypes of TRP channels, closely related to thermal sensation of organisms, in the ABI 7900 Realtime PCR instrument. Three functions of temperature control,signal capture and data process can be realized concomitantly within one assay system to establish in this subject. On this basis, biologically molecular mechanisms of twenty clinically effective Chinese herbs with natures of cold and hot will be explored in this project. This application is an original design and a profound study based on our previous work.The aim of this project is to solve the key technical and methodological problem which is preventing us from furthering our study on exploration of scientific connotation of the four natures of Chinese herbs. Implementation of this project will provide technical and methodological support for both uncovering biological significance and identifying active ingredients of the cold-hot natures of Chinese herbs.

我们课题组在完成国家自然科学基金过程中发现，中药寒热属性与热稳态调控网络中的关键节点-寒热感受TRP通道密切相关。而进一步的研究，亟需建立一套与寒热感受变化相适应的能够客观表征中药寒热属性与TRP通道关联特征的高通量生物学标准评测体系。基于此，本项目拟以与寒热感受关系密切的TRP通道亚型为关注点，以DRG神经元和HEK293细胞为载体分别构建原位和异源表达体系，在新型荧光定量PCR仪上搭建自动温控、钙离子荧光信号捕获、数据信息处理-三位一体的创新性技术平台。在此基础上，通过对寒热性中药的药性测试研究，挖掘寒热感受TRP通道与中药寒热属性的相关性和关联特征。该项目是一种全新的科研思路，是对前一个国家自然科学基金工作的深入和拓展，其目的是解决制约我们进一步诠释中药寒热药性科学内涵的瓶颈问题。本项目的实施将为深入揭示中药寒热四性的现代生物学基础提供强有力的技术支撑和实验积累。

本课题通过优化分离培养参数首先建立了TRP通道原位表达体系—背根神经节（DRG）原代培养细胞，同时基于慢病毒载体转染HEK293细胞构建了表达TRPM8、TRPV1和TRPA1通道异源表达体系，分别采用荧光定量PCR、Western blot、共聚焦等方法对HEK293细胞中TRP通道的表达进行了验证，并以TRP通道的激动和阻断等方式对通道的功能活性进行了评价，在新型荧光定量PCR仪上进行了测试分析研究。结果显示，TRP通道的原位和异源性表达体系功能良好，适于基于TRP通道的中药活性测试分析研究。在此基础上，项目组分别选择了临床上常用的10个寒凉性和10个温热性中药及其主要的活性成分进行了测试分析，结果表明，部分寒凉中药及其活性成分对TRPM8和TRPA1通道功能具有一定的上调作用，对TRPV1通道功能具有下调作用；部分温热性中药及其活性成分对TRPM8和TRPA1通道功能具有一定的下调作用，对TRPV1通道功能具有上调作用。原位和异源性体系相比，中药和活性成分对同一通道的作用具有明显的规律性，但不完全一致，可能与两种体系中的TRP通道源于不同种属有关。相比于原位体系，异源性TRP通道的表达体系更加均一稳定，更适于药物对TRP通道的作用和作用机制研究。本研究结果表明寒热性中药对TRP通道的调节作用具有一定的规律性，可能与其表征的寒热特征药性相关，为进一步全面揭示寒热药性的科学本质提供了一种新的方法，并积累了有效数据。