【作者】 张占军；

【导师】 王永炎； 王忠；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2005， 博士

【摘要】 本研究旨在通过对脑缺血后清开灵组分及其配伍治疗脑缺血基因表达谱变化及其药理学和药效学特色的研究,权衡组分配伍优劣,从分子水平提取有关功能信息的关键因素分析清开灵组分配伍的复杂体系,并且通过相关基因表达特征分析,为方剂配伍研究提供借鉴,为脑缺血药物筛选平台、药效评价方法以及现代中药药物配伍优化和中药资源的合理配置提供重要理论参考及依据,最大程度接近导师王永炎院士所提出的中医药研究需在整体论、系统论、理性论指导下才能更好阐发其原创优势的科学观和发展观。首先,用线栓法建立大鼠脑缺血再灌注模型。提取临床疗效较好的清开灵中的四个主要组分黄芩苷,栀子苷,胆酸,珍珠母作为研究对象。为展示方剂的综合效应,整体功能及组分功效的共性和个性特点,实验分为西药对照尼莫地平治疗组,模型组及黄芩苷,栀子苷,胆酸,珍珠母四个单一组分治疗组的同时,增加了黄芩苷+栀子苷,黄芩苷+栀子苷+胆酸,黄芩苷+栀子苷+胆酸+珍珠母,栀子苷+胆酸四个层次不同而又紧密相关同属一系统的组分配伍组。通过脑组织TTC染色测量药物治疗后脑梗塞面积,神经功能检查法对脑缺血后动物进行行为测评,病理及超微组织观察作为药效评价辅助手段。考虑到复杂性研究所遵循的整体、活体、动态观察原则,应用核磁共振弥散加权成像和弥散张量成像技术于造模后1h、脑缺血再灌注3h、12h、24h进一步综合评价模型是否成功及组分配伍的治疗作用,准确界定不同方剂组分配伍的功效层次。根据脑缺血后机体复杂的生理病理反应,简化复杂问题,从先期973工作基础及国外文献报道基因组数据库中提取了372个基因作为靶标,建立了涵盖脑缺血病生理反应特异表达基因及部分非特异表达基因在内的cDNA芯片。于脑缺血模型再灌注后3h、12h、24h分别提取每组3只鼠脑组织总RNA与芯片进行杂交,获取与治疗各组药效功效层次相应时间点对应的芯片杂交基因表达谱数据。采用管家基因(Housekeeping gene)校正和局部加权最小二乘法(LOWESS)方法对数据进行标准化校正,差异表达基因Ratio值和不同矩阵、不同芯片中同一基因的p值做为筛选具有显著统计性意义表达基因的标准。根据国际通用的GO分类原则,运用国际公认的Genespring软件对标准化后数据进行功能分类分析和聚类分析;通过脑缺血后不同组分差异表达基因的个性和共性特点及生物信息学相关研究,结合方剂配伍治疗脑缺血药效,中药对病理环节干预的偏重,权衡复杂体系中方剂配伍的利弊。通过荧光实时定量PCR,蛋白印迹以及指纹图谱特征分析方法对基因芯片实验技术平台及实验结果进行综合评估。基因表达谱权衡清开灵组分配伍治疗脑缺血药效特征分析研究 配伍组黄答普+桅子昔十胆酸+珍珠母,黄琴昔+桅子普+胆酸在TTC染色,行为学,形态学,磁共振成像研究中都体现了显著疗效,黄岑营+桅子昔,桅子昔+胆酸配伍组在所有药效评价指标中,也有一定的治疗作用,但治疗作用较前两组差。尽管黄答昔,桅子昔,胆酸三个单一组分在TTC染色脑梗塞面积测量中体现了治疗作用,但在动物行为测评及活体磁共振检查中均未见明显治疗作用。珍珠母在所有药效评价指标中均未见显著治疗效果,在四组分配伍中也没有起到药效叠加作用。在黄苏昔、桅子昔、胆酸、珍珠母四个组分治疗组中,共同差异表达的基因只有6个,每组与其它三个组分别作比较,差异表达基因数目显著不同,黄答昔治疗组有16个,桅子普治疗组有3个,胆酸治疗组为19个,珍珠母治疗组仅有1个。黄芹普+桅子普,桅子昔+胆酸,黄芹昔+桅子营+胆酸,黄芹昔+桅子营+胆酸+珍珠母治疗组的差异表达基因分别为65,24,53和62个,这些基因涉及到了信号转导、蛋白结合、转录调节等多方面。 通过不同组分及其配伍药效及其基因表达谱特征比较分析可以得出以下结论:中药复方配伍的治疗作用较单方效果好,凭借目前一、两种常用的评价方法,难以真正准确和全面地判定药物药效,现有的治疗脑缺血药物筛选平台及药效评价方法,需增加一些活体、动态、全面、综合的分析方法;在治疗脑缺血中,胆酸可以完全发挥珍珠母的药理作用并优于珍珠母,在我们目前开展的二者组方配伍研究中,珍珠母可以弃置不用,可见任何一种中药的任何一种效应,都是来自该中药的一种特定的中药分子组合,基因表达谱特征能够反应这种组合产生的特定效应;中药合理配伍可以起到减毒增效作用;中药配伍的增效作用机制可以通过启动新的作用途径和作用因子而实现;通过基因表达谱评判药效是可行的,组分配伍所产生的药效并不是依赖各单一组分作用及基因数目的叠加而实现的,更重要的是配伍后的整合作用,这种整合作用体现了中医中药作为复杂理论科学的原创优势和整体性,全方发挥疗效的整体作用机制同样在基因mRNA这样的微观世界得到体现;研究中医、中药作用机理,离不开整体观的指导,基因芯片技术是能较好体现或证实中医中药作为复杂理论学科“整体观”的原创优势和整体性及非线性特征的有效研究手段;这种整体综合评价方法的科学应用可以反证配伍优劣,优化配伍组合,指导调节这些非线性关系可以使药效达到最佳。 综上所述,本文在国内外首次将王永炎院士所提出的?更多还原

【Abstract】 In this research, we investigated the changes of gene expression profile and characteristics of pharmacology and pharmacodynamics of Chinese medicine composition and compatibilities against cerebral ischemia, weighted the advantages and disadvantages of composition compatibilities, attained key factors about functional information from the molecular level to analyze the complex system of prescription, and also analyzed the characteristics of related gene expression to provide important theoretical references for the drug screening platform of cerebral ischemia, pharmacodynamic evaluation, the optimization of Chinese medicinal materials compatibility and the reasonable collocation of their resource. Therefore, this research has approached to the fullest extent of science viewpoint and development viewpoint brought forward by tutor-Academician Wang Yongyan that the original advantage of traditional Chinese medicine can be explained better only when theresearch of traditional Chinese medicine directed by holism, system theory and rationalism.First, rat model of ischemia reperfusion was established with thread. We took the four main compositions of Qing Kai Ling, a prescription of good effects in clinic,----baicalin, jasminoidin, cholalic acid and concha margaritifera usta as research objects. To show the integrated effects and whole function of prescription, and commonness and individuality characteristics of composition effects, we divided the rats into Nimodipine control group, model group, the different 4 treatment groups of single composition----baicalin, jasminoidin, cholalic acid and concha margaritifera usta, additionally, the different 4 composition compatibility groups of different levels and closely related to onesystem----baicalin+jasminoidin, jasminoidin+cholalic acid, baicalin+jasminoidin+cholalic,baicalin+jasminoidin+cholalic +concha margaritifera usta. Area of cerebral infarction was measured through brain tissue TTC staining, ethology was measured through nervous function examination, and pathology and ultrastructure observation as the assistant methods of medicine effects evaluation. In view of the principles of holistic, vivid and dynamic observation, at the time points of 1h after operation, 3h, 12h and 24h after reperfusion, we applied vivid dynamic MRI of dispersed weight image and dispersed tension image to synthetically evaluate the effects of composition and their compatibilities, and to delimit the function level of different compositions and compatibilities.According to the complex physiological and pathological changes after cerebralischemia, we picked out 372 genes from gene bank as clone targets, and set up the cDNA chips including specific expression genes and partial nonspecific expression genes after cerebral ischemia. At the different time points of 3h, 12h and 24h after cerebral ishemia reperfusion, the total RNA of 3 brains from every group was hybridized with chip and get the data of chip hybridization gene expression profile corresponding to the different time points of function level in every group. The data were normalized through housekeeping gene correct and LOWESS method, ration value of the same gene in different matrixes and chips as the standard for screening the significant expression genes. Based on universal GO classified principle, we applied Genespring software to analyze the standard data for functional classify and clustering. Through study on the individual and common characteristics of different gene expression in different compositions after cerebral ischemia and biological informatics, combined with the effects on cerebral ischemia treating by prescription compatibility, the stress of Chinese medicine on pathological links, we weighed the advantages and disadvantages of prescription compatibility in complex system. The experimental technology platform of Gene chips and results were synthetically evaluated through the technologies such as fluorescence real time quantity PCR, Western blot and characteristic analysis of fingerprint.There were obvious effects in the com更多还原