实时荧光定量PCR技术及其在中医药研究中的应用

Application of Real-time Fluorescence Quantitative PCR in Chinese Medicine Research

摘要:

在中医现代化研究过程中, 越来越多地应用先进的现代科学技术, 聚合酶链式反应(PCR)技术作为一项重要的研究工具, 亦被不断地用于中医药的研究.荧光定量PCR是在PCR技术基础上发展起来的一种高度灵敏的核酸定量技术,在PCR反应体系中计入荧光染料或荧光探针,利用荧光信号积累实时监测整个PCR反应进程,最后通过标准曲线对未知模板进行定量分析,具有操作简便、快速高效,高通量而且高敏感性等特点,极大地克服了原有PCR技术的不足,目前该技术已逐渐成为中医药研究中的重要手段.本文概述了实时荧光定量PCR技术分别在中医诊断、中医药理研究、中医证实质研究和针刺作用机制研究中的应用.通过文献研究表明:应用实时荧光定量PCR,可以从分子基因水平探讨方剂或单味药及有效成分在组织、细胞及至分子层次上的作用机制,了解药物作用部位及靶点.探讨中医证候的病理生理学基础,并为阐明其生物学本质提供了新思路.使我国传统中医药体系的科学性在分子生物学的基因水平上得到了初步的验证.这些研究思路和方法为中医药的研究提供了借鉴,实时荧光定量PCR有望更广泛的应用于中医药研究中.

Abstract:

In the process of modernization research of traditional Chinese medicine, more and more advanced technologies are applied. Polymerase chain reaction (PCR), as an important research tool, is also used in the study of traditional Chinese medicine(TCM). Real-time fluorescent quantitative PCR is a highly sensitive nucleic acid quantification technique that is developed on the basis of technology of PCR. In PCR reaction system, fluorescent dyes or fluorescent probes are added; then the PCR reaction is monitored in real time by using fluorescence signal accumulation; and finally the unknown template is analyzed quantitatively by using standard curve. With the characteristic of simple, fast, efficient, high-throughput and high sensitivity as well, real-time fluorescence quantitative PCR overcomes the deficiency of traditional one greatly and gradually becomes an important means of studying the Chinese medicine at present. The applications in the diagnosis of TCM, pharmacology research of TCM, essence research of TCM syndrome and the study of acupuncture mechanism are introduced in this review. Many studies have showed: Molecular mechanism of the prescription, single herb and effective components at tissue, cell and molecular levels could be explored and target site of drug could be found by using real-time fluorescent quantitative PCR; Additionally, it helped to reveal the pathophysiological basis of TCM symptoms and provide new ideas for elucidating the biological essence; What's more, the science of traditional Chinese medicine has been validated at the genetic level of molecular biology. Based on these studies, real-time fluorescent quantitative PCR would be widely used in the research of traditional Chinese medicine.