【作者】 郑一；

【导师】 张学文； 尹岭；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2004， 博士

【摘要】 1 目 的 中风病是中老年人的常见病, 多发病, 严重危害人类健康. 本课题的目的是讨论颅脑瘀水毒邪与缺血性中风病发病的关系和脑络舒通治疗缺血性中风的药效学机制. 本课题在中医基础理论的指导下 ,分析了缺血性中风的发病规律, 认为瘀, 水 ,毒邪互结于脑内是中风病急性期的病机重点. 提出活血利水 ,解毒通络治法是缺血后脑水肿治疗的关键. 从脑络舒通对脑组织含水量, 血脑屏障通透性-EP和MMP-9等方面深入探讨脑络舒通的利水作用, 采用血瘀证大鼠凝血指标和临床中风病人血瘀证证侯积分的方法证明脑络舒通的活血功能 .通过观察临床病人血清TNF-α和IL-1的变化来探讨脑络舒通的解毒作用,并从bFGF和nestin 蛋白的表达来讨论脑络舒通对成体神经功能重塑的治疗机制. 2 方 法 2.1 采用光化学诱导大鼠局灶性脑缺血的方法, 设脑络舒通治疗组 ,模型对照组 ,假手术组, 空白组 ,于造模成功后3h始,给予脑络舒通灌胃,连续4d 动态观察体重及神经功能评分的变化,术后第5d 取脑TTC染色后计算梗塞灶体积HE染色后观察病理学改变. 2.2 采用栓线法造成大鼠大脑中动脉缺血3h, 再灌注24h 模型 ,观察各组神经功能评分脑组织含水量 ,用 Evans 蓝测定法观察血脑屏障通透性的改变 ,并采用免疫组织化学方法观察缺血再灌注大鼠大脑-EP表达. 采用RT-PCR方法测定MMP-9mRNA 的表达. 2.3 采用大鼠血瘀模型,观察脑络舒通对血瘀证大鼠凝血,纤溶系统的影响.并在临床观察脑络舒通治疗前后治疗组和对照组病人的血瘀证证侯积分的改变. 2.4 采用放射免疫RIA法观察脑络舒通治疗前后治疗组和对照组病人血清TNF-α 和IL-1 的变化. 2.5 采用免疫组化方法观察光化学诱导的脑缺血大鼠治疗组, 模型组, 假手术组, 空白组脑的第5d 时bFGF的表达和第10d 时nestin 蛋白的表达. 3 结 果 3.1 脑络舒通可以减小光化学诱导的大鼠局灶性脑梗死体积, 治疗组梗死灶体积为7.322.03mm3 明显小于对照组(16.01 2.62mm3)( P<0.001).治疗组大鼠的缺血性病理改变比对照组明显减轻,假手术组和空白组无缺血灶形成. 3.2 MCAO大鼠脑缺血3h再灌注,24h的对照组缺血侧脑组织含水量(79.24 0.61%)和EB含量( 4.78 0.72ug/g) 明显高于空白组(73.43 0.57%)( 2.07 0.15ug/g)治疗组缺血侧脑组织含水量(76.85 0.87%)和EB含量( 3.52 0.63ug/g )明显降低 ,与对照组相比具有显著性差异( P<0.001 )假手术组与空白组则无显著性差异.对照组非缺血侧脑组织含水量(75.590.48%)和EB含量3.24 0.57ug/g 也较空白组增高(P<0.001). MCAO大鼠缺血3h 再灌注24h 时,对照组大鼠缺血灶 -EP 的表达明显升高,以神经胶质细胞表达为主,治疗组大鼠缺血灶 -EP 的表达与对照组相比明显减少.具有显著性差异(P<0.001) 假手术组与空白组相应缺血灶-EP的表达较少,无显著性差异( P>0.05) MCAO大鼠脑缺血3h 再灌注24h时,对照组大鼠(MMP-9mRNA)的表达明显升高,与假<WP=5>手术组和空白组相比具有显著性差异(P<0.001),治疗组大鼠(MMP-9mRNA)的表达较对照组减少(P<0.05).假手术组和空白组大鼠表达较少, 相比较无显著性差异. 3.3 血瘀证大鼠凝血-纤溶指标观察中, 血瘀证大鼠与空白组比较, PT, APTT 明显缩短.(P<0.01 P<0.001) Fig.显著增加(P<0.001), PA 显著增高 (P<0.001).说明血瘀证大鼠存在高凝状态, 治疗组PT, APTT 较对照组延长(P<0.01) Fig 和PA 降低(P<0.001). 3.4 对照组病人血清, TNF- 含量在治疗后 7d 312 041ng/ml 略有下降 与入院时351 045ng/ml 相比无显著性差异(P>0.05) 治疗后 21d血清TNF- 含量 2.810.59ng/ml 与治疗前相比具有显著性差异 (P<0.01) . 治疗组病人血清TNF含量在治疗后7d 2.39 0.33ng/ml 迅速下降 治疗后21d 血清TNF- 含量 1.77 0.48ng/ml 明显下降P<0.001 与对照组相比具有显著性差异(P<0.001) 两组治疗后血清 IL-1 含量较治疗前下降 P<0.05 治疗组下降 0.38 0.16ng/ml 较对照组 0.52 0.12ng/ml 明显 具有显著性差异 P<0.001. 3.4 光化学诱导脑缺血第5d时, 治疗组大鼠缺血边缘区 bFGF 的表达明显增高 ,神经元细胞和神经胶质细胞均有表达 ,与对照组相比具有显著性差异(P<0.001).治疗组海马锥体细胞层中bFGF的表达明显增高, 与对照组相比具有显著性差异(P<0.05) 假手术组与空白组相应缺血边缘区及海马没有bFGF的表达 .各组缺血灶内部 ,皮层与皮层下, 小脑等部位均无bFGF 阳性细胞的表达. 光化学诱导脑缺血第 10d 时, 治疗组大鼠侧脑室室管膜下层和第三脑室底部 nestin 蛋白的表达明显增高, 与对照组相比具有显著性差异( P<0.001 ),假手术组与空白组侧脑室室管膜下层和第三脑室底部仅有少量nestin蛋白表达. 治疗组大脑皮质缺血边缘区 nestin 蛋白表达明显增高. 与对照组相比具有显著性差异( P<0.05) 缺血区周围的大脑皮质和皮层下nestin蛋白的表达两组无显著性差异, 假手术组与空白组无阳性细胞表达. 4 结 论 4.1 脑络舒通可减轻MCAO鼠缺血再灌注对血脑屏障的损伤.对血脑屏障通透性具有一定的保护作用, 可抑制脑内 -EP 的合成和释放. 降?更多还原

【Abstract】 1 Objectives Stroke is the most common disease in the aged, which has damaged the health of human beingseriously. The thesis discussing the relatively aspects pertain to the relation between the stasis,waterand toxic and IVCD, and the pharmacodynamic mechanism of NLST in the develepmenting ofIVCD. This topic guided by the rational of TCM analysised the episode regularity of ischemic stroke,and realized the collection of stasis, water and toxic in the brain is the focus pathology in the acuteperiodofstroke,andgavea suggestionthatbloodactiviting diuresis,detoxicate is the keywayinthetreatment of cerebral edema caused by ischemia. It discussed the diuresis function of NLST from itseffects on the water volume in the brain tissue blood brain barrier permeability neuropeptide andMMP. And discussing the detoxication of NLST by observing the changes of TNF-α IL-1 in theblood serum of patients and therapeutic mechanism of NLST in the nerve function remodeling fromthe exprssion of neurotrophic factor and proliferation of nerve stemcell. 2 Method 2.1The focal cerebral ischemia model was induced by photochemistry in rats, all experimentalrats were divided into NLST treatment group, model control group, pseudoperarion group, vacuitygroup .NLST was successively poured into rats’ stomach for 4days begun in 3 hours after cerebralischemia was induced. Rats’ weight and clinical score were detected each day. The brain wereexcised in the 5th day of postoperation, HE stain and TTC stain were done for measuring volume ofinfarction and observing pathologic change.2.2 By adopting MCAO in rat models, achieve MCAO3h/R24h, observing nerves function scoreand water volume in the brain tissue of each group. Observing BBB permeability variant by themethod of Evans blue fluorometry, and Observing cerebrum -EP Protein Expression of processedMCAO/R rats by the method of immunohistochemistry, Estimate the expression of MMP-9mRNAby RT-PCR. 2.3 By constructing rats blood stasis model, and analyse blood, observe the affect of NLST tothe blood coagulatlon-Fibrinolysis systemof blood stasis rats. 2 4 To make RIA by liquid phase competition, Observe the influence of TNF-α and IL-1 inthe patients’ blood serum between treatment group and contrast group in clinical pretherapy andpost-treatment by NLST. 2 5ObserveExpressionof5thdaybFGFand10thdaynestinproteinoffocalcerebralischemiainduced by photochemistry in rats of NLST treatment group, model control group, pseudoperariongroup, vacuity group by immunohistochemistry. 3 result 3 1 NLST can reduce the volume of infarction of rats induced by photochemistry. The volume<WP=8>英文摘要 5of infarction of treatment group was 7.32±2.03mm3, the volume of the contrast group is16.01 2.62mm3, a sharp contrast can be observed between the two group the volume of infarction(p<0.001). thepathologic change of treatment group is relieved than constract group after the drug. 3 2 MCAO3h/R24h in rats, water volume and EB content in the brain tissue in the side ofischemia ofcontrol group(79.24 0.61%) 4.78 0.72ug/g ,whichhavea higher volumethanthevacuity group(73.43 0.57%) 2.07 0.15ug/g ,water volume andEBcontentinthebraintissueinthesideofischemiaofTreatmentgroup(76.85 0.87%) 3.52 0.63ug/g issignaificantlyreduced,have a significant difference compared to model control group(P<0.001). pseudoperarion group hadno significant difference comparedto vacuity group. water volume and EB content inthe brain tissueinthe sideofnon-ischemiaofcontrl group(75.59 0.48%) 3.24 0.57ug/g isalsohigher thanthevacuity group(P<0.001). MCAO3h/R24hinrats,theexpressionof -EPproteininthebraintissue intheside ofischemiaof model control group obviously increased, expressed by glial cell primary. the expression of -EPprotein in the brain tissue in the side of ischemia of treatment group is decreased obviously, have asignificant difference compared to contr更多还原