【作者】 李莹；

【导师】 程翼宇；

【作者基本信息】 浙江大学 ， 中药学， 2008， 硕士

【摘要】 蛋白质组学的诞生与发展,为研究蛋白质表达和功能变化提供了强有力的技术支持,使人们可以从系统的角度了解药物在蛋白质水平上对机体的作用。中药具有多组分、多靶点、整合调节的作用特点,运用蛋白质组学技术发现中药活性物质可能的作用靶点,了解中药作用途径,将有助于在分子层次上诠释传统中医药理论的现代生物学意义。本文以急性心肌缺血大鼠的心肌蛋白质组为研究对象,首先通过实验研究建立和优化心肌蛋白质组的双向电泳分析方法;然后利用该方法对芪参益气方的抗急性心肌缺血作用机制进行研究,主要研究内容如下:1.大鼠心肌蛋白质组分析方法研究:从大鼠急性心肌缺血模型中获取缺血区心肌组织,研究建立蛋白提取、纯化等方法。通过对心肌蛋白质组分析过程中各关键参数的优化(包括上样量,IPG胶条规格,等电聚焦条件和SDS-PAGE电压等条件),建立了心肌蛋白质组学双向电泳分析方法。运用该方法对维拉帕米作用下急性心梗大鼠的心肌蛋白组进行分析,结果表明该法能较理想地分离出差异蛋白,并获取了15个差异蛋白进行质谱鉴定。2.芪参益气方抗急性心肌缺血作用的蛋白质组学研究:对中药方剂芪参益气方抗心肌缺血作用开展药效学实验,并利用已建立的方法对芪参益气方给药3天与7天的心肌缺血大鼠心肌蛋白质组进行分析,研究发现35个蛋白差异点。对这些差异表达的蛋白进行质谱鉴定后发现其与能量代谢、抗氧化应激等作用有关。研究结果表明,芪参益气方的心肌保护作用与恢复心肌损伤过程中的能量供应、对抗氧化应激等作用有关。通过以上研究,本论文成功建立了心肌蛋白质组学的实验研究方法,利用该法对芪参益气方的抗急性心肌缺血作用进行了蛋白质组学研究,研究结果为揭示该方剂多途径作用机制提供了一定的线索。更多还原

【Abstract】 Proteomics is a new emerging technique, which offers a systematical view on the variation of expression and function of protein under certain stimuli. It helps people get new insight of drug action on the proteo-level. The traditional Chinese medicine has the characteristic of the multi-component, multi-targets and synergistic effects. Using the proteomic technique to identify the potential mechanism of traditional Chinese medicine will contribute to understand the way that the traditional Chinese medicine works by. It will also benefit to reveal modern biological meaning of the theory of traditional Chinese medicine on the molecular level. In this thesis, cardiac proteome of the acute myocardial ischemic rats were studied. A two dimensional electrophoresis (2-DE) based proteomic analytical method was developed by a series of comparative experiments. Subsequently, the cardioprotective effects of Qi-Shen-Yi-Qi formula in- acute myocardial ischemic rats were studied by the routine pharmacological assays and cardiac proteome analysis. The main research contents were as followed:1、Firstly, a 2-DE based method for the analysis of rat myocardial proteome was established. The tissue of cardiac muscle were obtained from the rats with acute myocardial ischemia. The method of myocardial proteomic sample preparation (include protein extraction and purification) was developed. Several key parameters in 2-DE analysis were optimized, including sample amount, IPG strips, voltage for focusing and SDS-PAGE. The established method was applied in analyze proteome of ischemic hearts being treated with verapamil. 15 differential proteins were well separated. The results indicated the present method was reliable and stable, which can be used to characterize myocardial proteome.2、The proteomic study on cardioprotective effects of Qi-Shen-Yi-Qi formula in acute myocardial ischemic rats. The pharmacological assays were performed to determine serum enzyme levels in each experimental group. The proteomic analysis was also progressed by the built 2-DE method to rats cardiac proteome which was treated with Qi-Shen-Yi-Qi formula for 3 and 7 days after heart ischemia. The result showed that the expression of 35 proteins were changed significantly. Using MALDI-TOF-MS, these 35 proteins were identified by database searching. These proteins can be related to energy metabolism and mitochondrial function, anti-oxidative stress property, and so on. The findings showed that the myocardial protective effects of the qishenyiqi formula in myocardial damage process are related to the recovery of energy supply as well as anti-oxidative stress property.In this thesis, the method of myocardial proteome was built successfully, and the proteomic research on the anti-ischemic effect of the Qi-Shen-Yi-Qi formula was progressed by this built method, the result afforded some clues in multiple targets mechanism of the traditional Chinese medicine.更多还原