论文标题:从急性脑缺血再灌注炎症因子动态变化探讨内毒的形成及作用
论文作者
论文导师 张允岭；王少杰；白文,论文学位 博士,论文专业 中医内科学
论文单位 北京中医药大学,点击次数 71,论文页数 70页File Size1555K
2007-05-01论文网 http://www.lw23.com/lunwen_51373517/
interleukin-1β;; cerebral ischemical reperfusion injury;; endogenous toxicmaterials;; tumor necrosis factor-α
缺血性脑血管疾病是威胁人类健康与生存的主要疾病之一,是中老年人常见病,具有高发病率、高患病率、高死亡率、高致残率、高复发率的特点,是目前重点防治的一种疾病。既往在因于风、因于火、因于痰、因于瘀等的认识基础上,采用中医单一或多因的辨证论治,取得了一定的疗效,但进一步的疗效提高实在艰难。近年来,随着对传统毒邪认识的深化,中风病临床实践的发展和现代病理机制研究的深入,提出从毒论治中风病,从而提高中风病疗效,已成为中风病病因学及治疗学研究中新的视点与热点。内毒由脏腑失和,气血不调,风、火、痰、瘀等内生诸邪炽盛或久郁不解,相生互结互化,酝酿而成,并可合而为患,危害更甚。可引起机体功能破坏、丧失和/或败坏形质、导致病情恶化加重或呈沉疴状态并难以干预。 因此,我们在中风病内毒理论的指导下,以局灶脑缺血炎症反应为切入点,研究了脑缺血再灌注不同时间点炎症因子的动态变化以及其蛋白、基因的水平,来探讨内毒与炎症因子的关系,进一步研究内毒的产生和作用过程。 研究目的: 1.从局灶脑缺血再灌注炎症因子的动态变化探讨内毒形成及作用。 2.探讨具有清热解毒活血开窍作用的清脑宣窍滴丸是否通过抑制急性脑缺血炎症反应减轻急性脑缺血损害。 3.通过急性缺血级联反应过程中炎症因子、细胞间黏附分子对神经细胞的“毒性效应”阐释内毒的本质是损伤和败坏脑组织,并具有不可逆转性,是中风病难治难愈的关键原因和核心病机,从而为中风病内毒学说找到新的理论支持;同时赋予“解毒通络”法新的生物学内涵。 研究方法: 1.采用线栓法制备大鼠左侧大脑中动脉脑缺血再灌注模型,缺血1.5h,分别再灌注1h、3h、6h、12h、24h、48h,用免疫组织化学方法测定上述各时间点TNF-α、IL-1β,并进行组织病理学观察。 2.采用线栓法制备大鼠左侧大脑中动脉脑缺血再灌注模型,缺血1.5h,再灌注12h后断头取脑,于冰盘上迅速取缺血侧大脑半球,制备10%脑组织匀浆,用ELISA方法测定患侧脑组织TNF-α、IL-1β、ICAM-1的含量。 3.采用线栓法制备大鼠左侧大脑中动脉脑缺血再灌注模型,缺血1.5h,再灌注12h后选取皮层组织进行总RNA提取和TNF-α、IL-1βmRNA的表达。 研究结果: 1.模型组大鼠均出现进食量减少,精神状况差,活动减少、倦卧,被毛无光泽,体重均明显降低。 2.假手术组大鼠无神经功能损伤的症状,行为学评分为0分;模型组大鼠表现为对侧前爪内收、向对侧转圈行走、向对侧倾倒等,以缺血再灌后12h最为显著;中药组表现以对侧前爪内收、向对侧转圈行走为主,神经功能缺损较模型组减轻,二者有显著差异(P<0.05) 3.假手术组大鼠大脑皮质层次结构清晰,模型组各组皮质均可见细胞肿胀,胞浆嗜酸性变,胞核深染、碎裂、溶解、固缩或消失,胶质细胞呈空泡样变性;锥体细胞形态欠规则,神经细胞呈三角形或多角形。脑缺血再灌注24h上述变化明显,清脑宣窍滴丸治疗组上述变化减轻。 4.模型组大鼠TNF-α缺血再灌注3h后即有阳性表达,24h达到高峰,其后逐渐下降,其中再灌注6h、12h、24h、48h组和假手术组比较有显著性差异(P<0.01);模型组大鼠IL-1β缺血再灌注6h后即有阳性表达,12h达高峰,随之开始逐渐下降,其中再灌注6h、12h、24h、48h组和假手术组比较有显著性差异(P<0.01)。 5.大鼠脑缺血1.5h再灌注12h后,脑组织中TNF-α、IL-1β、ICAM-1的含量与假手术组比较均明显升高,其差别有极显著性意义(P<0.01)用药组和模型组比较均有不同程度的降低(P<0.05或P<0.01)。 6.大鼠脑缺血1.5h再灌注12h后,模型组TNF-α、IL-1βmRNA的表达与假手术组比较均明显升高,清脑宣窍滴丸治疗组表达下降。 研究结论: 1.急性脑缺血再灌注后,模型组大鼠病变侧脑组织中炎症因子TNF-α、IL-1β表达明显升高,表现出不同的时程规律;它们的峰值集中在再灌注后12h-24h这一段时间,表明TNF-α、IL-1β参与了脑缺血病理损害,是脑缺血级联反应的重要环节,与急性脑缺血后神经细胞损害的发生和程度密切相关,其动态变化和毒性效应体现了内毒形成及作用过程。 2.急性脑缺血再灌注12h后,和假手术组相比模型组大鼠TNF-α、IL-1β、ICAM-1蛋白和mRNA均增高,应用清脑宣窍滴丸干预后均有不同程度降低,表明清热解毒活血开窍作用的清脑宣窍滴丸可能通过抑制急性脑缺血炎症反应减轻急性脑缺血损害。 3.急性缺血级联反应过程中炎症因子、细胞间黏附分子对神经细胞的“毒性效应”与中医学中内毒形成及作用过程具有相似或相同之处。这些毒性物质共同作用的后果引起或加重神经细胞坏死或凋亡、脑组织损害;体现了内毒的本质是损伤和败坏脑组织,并具有不可逆转性,是中风病难治难愈的关键原因和核心病机。 4.炎症因子、细胞间黏附分子与急性脑缺血损害的关系可以为中风病内毒学说找到新的理论支持;同时赋予“解毒”法新的生物学内涵。
Ischemic cerebrovascular disease is one of the important diseases that threaten the healthand life of humanbeings,and it also is the common disease in aged people,high in attact rate,death rate and mutilated rate. Base on the causes of this disease in Chinese Medicine as wind,fire, sputum or stagnation , and the determination of treatment based in pathogenesis obtainedthrough differentiation of symptoms and signs, we have gained some therapeutic effect.However, it is difficult to improve the therapeutic effect and clinical repeatability. For the pastfew years, along with the propose and advance of toxicant theory , and the deeper research ofits pathomechanism, it has formed a new viewpoint and focus on treating stroke throughcontrolling toxicant. Endogenous toxic materials is caused by disharmony of entrails , or dyscrasia of qi andblood, or wind, fire, sputum, stagnation and so on. All these endogenic pathogenic factorsinterties and interconvert and make organism disfouncsion and quality nature corruption, itaggravate the patient’s condintion and so it is hard to interfere in. So, by the conduction ofendogenous toxic materials theory, we had a study on dynamic change of inflammatory factorsand their gene level on cerebral ischemia reperfusion rats to get the relationship betweenendogenous toxic materials and inflammatory factors so to understand how endogenous toxicmaterials generate and how they do harm to patients. Objective 1. To observe the formation of endogenous toxic materials and its effective coursethrough observing dynamic change of inflammatory factors in focus cerebral ischemiareperfusion rats. 2. To observe if the clearing brain and releasing orifices dripping pills with heat-clearingand detoxicating effect could lessen the acute cerebral ischemia damage through inhibiting theexpression of inflammatory factors. 3. To explain the essence of endogenous toxic materials is to damage the brain tissuethrough explaining the poisoning effect of inflammatory factors and adhesion molecule to thenerve cell, and prove the effect is inreversible, It is the most important cause that stoke isdifficult to be cured. This provide a new theoretical support to the endogenous toxicant theoryof stroke, and endue“detoxication method”with a new biological connotation. in themeanwhile. Methods 1. Suture-occuluded method was used to block the left middle cerebral artery to makeischemia for 1.5h, after reperfusion for 1h, 3h, 6h, 12h and 24h , the Zea longa method wasused to evaluate the neurologic impairment and immunohistochemistry method was used to detect the content of TNF-α, IL-1βin brain tissue, observe pathologic change in tissue at thesame time. 2. After reperfusion for 12h, get the cerebral hemisphere which was made ischemia on iceplate after decapitation and prepare 10% tissue homogenate. ELISA method was used to detectthe content of TNF-α, IL-1 and ICAM-1. 3. After reperfusion for 12h, get cortex issue and abstract the total RNA and detect theexpression of TNF-αmRNA and IL-1βmRNA. Results: 1. Rats in model group appeared to be inactivity, fewer food-intake, poor spirit conditionand weight losing. 2. Rats were lack of the symptom of neurologic impairment in sham operated group andshowed severe neurologic impairment in model group and, moreover, in treated group they gotmuch less neurologic impairment than model group.(P<0.05) 3. Disorganization of structure layer of cerebral cortex is severe after reperfusion for 12h,pathological change in tussue showed that nerve cell swelling, and there was fragmentation,dissolve, pyknosis or disappear of cell nucleus which reach to top after reperfusion for 24h,while cortex structure of sham operated group was clear and those changes in treated groupwas much less.. 4. TNF-αin model group express after reperfusion for 3h, and reach to top after 24h,then decreased gradually. IL-1βin model group express after reperfusion for 6h, and reach totop after 12h, then decreased gradually. Both had statistical significance compared withsham operated group after reperfusion for 6h, 6h, 12h, 24h, 48h. 5. After reperfusion for 12h, the content of TNF-α, IL-1β, ICAM-1 in model groupwere much higher than those in sham operated group（P<0.01）.After treated with clearingbrain and releasing orifices dripping pills, all those factors decreased(P<0.05 or P<0.01) 6. After reperfusion for 12h, the expression of TNF-α, IL-1βmRNA increased in modelgroup compared with sham operated group , and decreased in treated group. Conclusion 1. After acute cerebral ischemia reperfusion, inflammatory factor such as TNF-α, IL-1βinmodel group obviously increased , showed a time-related change. Their peak value wasbetween 12h-24h, that proved they take part in the important course of cascade reaction ofcerebral ischemia reperfusion and was the cause of pathological lesion. Inflammatory factorssuch as TNF-αand IL-1βclosely related to the impairment of nerve cell, The dynamic changeand poisoning effect of them could explain the mechanism of formation of endogenous toxicmaterials and how they take effect. 2. After reperfusion for 12h, TNF-α, IL-1β, ICAM-1mRNA and expression of theirproteinum increase in model group compared with sham operated group and decrease intreated group, that proved clearing brain and releasing orifices dripping pills with heat-clearingand detoxicating effect could lessen the acute cerebral ischemia damage through inhibiting the inflammatory reaction. 3. The poisoning effect of inflammatory factors and adhesion molecule in course ofcascade reaction in acute ischemia is similar with the formation and effected course ofendogenous toxic materials in Chinese Medicine. These poisoning materials take the totaleffect and induce or aggrevate the cell necrosis or apoptosis and damage brain tissue in the end.That proved the essence of endogenous toxic materials is to damage the brain tissue,moreover , it is inreversible. This is the most important cause that stoke is difficult to be cured. 4. The relation between inflammatory factors and adhesion molecule provide a newtheoretical support to the endogenous toxicant theory of stroke, and endue“detoxicationmethod”with a new biological connotation in the meanwhile.
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