【作者】 青雪梅；

【导师】 李澎涛；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2006， 博士

【摘要】 中风病因其高发病率,高死亡率,高致残率,而成为医药界的重点攻克项目。中医在中风病认识及治疗上有几千年历史,近年来,在临床及实验研究方面已取得了可喜成就,但仍有许多问题亟待进一步解决。王永炎院士认为,以毒邪和病络作为深入研究的切入点,是进一步提高脑血管疾病疗效的突破口所在。现代研究已认识到血管内皮不仅仅是血液与组织间物质交换的屏障,而且是一个重要的内分泌、旁分泌器官,具有强大的分泌、代谢功能。与既往的脑缺血离体实验中多以神经细胞为研究目标不同,我们以脑微血管内皮细胞(CMEC)做为研究的切入点,从一个全新的角度研究药物防治缺血性脑损伤进行了有意义的探索,对于指导脑系疾病的中医药现代化研究具有重要启示。本课题研究用药为通络救脑(TLJN)注射液,是治疗缺血性脑病的中药新药。已完成验收的“十五”国家攻关课题“通络救脑注射液临床前研究”在保护大鼠脑缺血组织损伤中获得了优良效果,并在“863”项目支持下的临床试验进一步肯定了其抗缺血损伤的效应。我们从“络脉”功能调控入手,以神经血管单元(NVU)做为一研究体系,从细胞、分子及基因水平进一步研究TLJN注射液“解毒通络”的药效机制。以脑微血管内皮细胞做为研究的切入点和枢纽,开展了脑微血管内皮细胞调控机制的药理学研究,旨在从理论与方法学两方面对现有的脑系疾病防治研究进行有意义的补充和积极的探索。实验方案共分三个部分:第一部分:系基础研究,原代培养大鼠脑微血管内皮细胞(rCMEC),并做氧糖剥夺(OGD)缺血模型,观察了TLJN注射液对其的保护效应及可能的黏附机制研究。第二部分:收集正常、缺血及药物干预的最佳时段脑微血管内皮细胞条件培养液,分别作用于正常、缺血及缺血再灌的大鼠皮层星形胶质细胞和皮层神经元,研究生理及病理状态下,内皮细胞分泌的活性物质介导的神经元及星形胶质细胞的生物学功能改变,探讨脑微血管内皮细胞与神经血管单元(NVU)中另外两种脑细胞的功能联系。第三部分:通过正常、缺血及TLJN干预的脑微血管内皮细胞的基因芯片初探及部分差异基因的进一步验证,从整体基因表达水平来研究缺血对脑微血管内皮细胞的影响及药物对其的调控作用,进一步深化“毒损脑络”病机理论的生物学内涵,诠释TLJN“解毒通络”的微观作用机制全貌。研究结果:1.成功地培养和鉴定了大鼠脑微血管内皮细胞,扫描电镜观察可见内皮细胞有丰富的微绒毛,透射电镜观察可见典型的紧密连接结构,免疫组化对Ⅷ因子鉴定,其纯度可达98%。2.TLJN注射液对正常及缺血脑微血管内皮细胞具有双向调节作用,药物剂量与细胞增殖活性呈非线性关系。重复三次实验,可以肯定TLJN小剂量范围(0.5～8μL·ML-1)均促进了缺血脑微血管内皮细胞活性,有统计学意义(P<0.05或P<0.01)。3.脑微血管内皮细胞缺血6h时,E-选择素和VCAM-1的表达均上调,提示CMEC缺血时启动了黏附机制。TLJN注射液干预缺血CMEC,能够下调E-选择素(P<0.01),更多还原

【Abstract】 Stroke is becoming a leading captured item in the domain of medicine because of its high incidence, high mortality, high deformity-causing. There is a history of several thousands years in understanding and curing stroke. Recently, delightful achievements have been acquired from the aspects of clinic and experiment, but a lot of problem is still desiderated. There should be a durance in bringing into predominant play in Chinese medical clinic and international spread due to short of arguable and repetitious quality in pathogenesis of stroke and ambiguous functionary mechanism of Chinese medicine. So it is imminence need to demonstrate pathological mechanism of stroke which is cured with Chinese medicine in modern science way. It is a Chinese medicine modernized way to dredge effect Chinese medicine from traditional medicine and prove its substance basement in experiment way. Academician Wang Yongyan put forward removing toxin to dredge brain collaterals that is meaningful in the combination of Morden Medicine and Traditional Chinese Medicine (TCM).In Chinese medicine, removing toxin to dredge brain collaterals as research key point is a break through to improve curative effect of cerebrovascular disease. It is realized that cerebral microvascular endothelial cells(CMEC) is not only a substance exchange barrier of blood and tissue, but also an important incretion apparatus. Our research pay more attention to CMEC rather than neurons, which is a meaningful probe to guide TCM realization mordernation used in brain disease.This study of drug is TongluoJiunao(TLJN) injection which is a traditional Chinese medicine employed in cerebral ischemia. The National Key Program of“pre-clinical research of TLJN injection”which has passed the test acquired outstanding effect in protecting rat injured tissue of brain ischemia. And its anti-ischemia damage effect has been further proved in clinical tests which was supported by“863”Research Projects. With study of CMEC as key target and NVU as a research system, we research pharmacologic mechanism of TLJN injection in cell, molecule and gene levers. The aim of this research is a meaningful supplement and an active probe of prevention and cure brain disease in theory and method aspects.Experiment is divided three parts:更多还原