【作者】 杨忠华；

【导师】 许能贵；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2009， 博士

【摘要】 脑血管疾病是目前严重危害人类健康的主要疾病之一,在我国死于脑血管病者多于心脏病及癌症,居三大死因之首。缺血性脑血管病是威胁人类的常见病、多发病,因此寻找有效的治疗手段并阐释其机理仍然是目前脑科学领域的研究热点。近年来在脑血管病的研究中发现卒中后康复的不同阶段,大脑功能和组织结构可以发生一定的自我修复和重建能力。在脑缺血的几分钟至数月大脑皮质出现明显的功能和结构变化,不仅发生在病灶周围,而且在远隔部位,这种可塑性称为脑的可塑性。缺血性脑损伤是神经损伤中最常见的类型,而突触是神经元之间信息传递和加工的部位,突触的功能与结构可随外界刺激或环境因素的影响发生明显的可塑性变化,因此是活动依赖性脑可塑性变化的关键结构部位。细胞外调节蛋白激酶(Extracellular signal regulatedkinase,ERK)是丝裂原活化蛋白激酶(Mitogen activated protein kinases,MAPK)的一种,是非常保守的信号系统,它们存在于从酵母到哺乳动物的各种细胞中。现在对于ERK的研究比较清楚,ERK调节着细胞的增殖、分化和存活,一方面接受大量来自生长因子、丝裂原、环境刺激等的信号,另一方面通过ERK信号级联反应作用于核转录因子,调控基因表达。目前,已开始寻找针对ERK途径中各个环节的调控机制,用以调控信号转导的途径,从而达到治疗疾病的目的。目的用科学的方法探索MAPK/ERK通路与脑缺血后神经元损伤的相关机理、相关特征、相关因素,相关规律,及电针干预的影响作用,为针刺治疗脑血管病及改善大脑功能的可塑性研究提供新的依据。方法选用SPF级雄性SD大鼠90只,由抽签法随机分为假手术组、模型组和电针组,其中假手术组、模型组和电针组每组又分为2h(小时)、1d(天)、3d(天)三个时间段组,共9小组,每小组10只大鼠。模拟临床脑梗塞的证型,选择以大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)为模型,采用临床常用的、对脑缺血及其后遗症有确定疗效的针刺和电刺激结合起来的电针治疗局灶性脑缺血。针对ERK通路在脑缺血中作用途径、作用机制这一关键问题,从多环节、多层次、多靶点上观察电针对ERK通路的影响及电针对脑缺血后海马神经元的保护作用,采用“百会”、“大椎”两穴,从海马CA1区和CA3区神经元入手,采用免疫组化及免疫荧光的方法来观察与脑缺血及突触可塑性密切相关的ERK1/2和NMDAR1、AMPA(GluR1)等受体的动态变化在累加电针后的表达,并通过采用Y迷宫实验测试大鼠的学习记忆能力以及神经行为学评分对大鼠造模及针刺干预结果进行客观评价,从而对针刺促进保护脑缺血后海马神经元和MAPK/ERK通路在脑缺血中的作用途径、作用机制及脑缺血早期突触可塑性的可能作用机理进行深入的探讨,为今后进一步阐明大脑可塑性的神经生物学机制以及为临床更好改善脑缺血后大脑功能的恢复提供更为确切的理论依据。结果一、神经体征评分数据通过spss13.0统计分析,方差齐性检验,P＞0.05(P＜0.05采用Dunnett分析),采用LSD和S-N-K检验法分析。假手术组大鼠神经行为学表现均正常,评分均为0分。大鼠的神经学评分显示,模型组3组之间两两比较均无统计学意义(P＞0.05),而针刺组1d组和3d组比较也无统计学意义(P＞0.05),但电针1d组和3d组分别与电针2h组比较均有显著差异(P＜0.01),有统计学意义,且电针1d组和模型1d组(P＜0.05)、电针3d组和模型3d之间(P＜0.01)比较也存在差异,有统计学意义。结果表明:模型组与电针组与假手术组比较,差异有统计学意义(P＜0.01),说明MCAO模型复制成功,造模导致大鼠脑组织的损伤,并且引起了相应神经功能缺失。电针组与模型组比较(除2h组),均有差异,存在统计学意义(P＜0.05),表明早期电针治疗对脑缺血在改善神经行为学方面有效果。电针组内1d和3d组无明显差异,没有统计学意义(P＞0.05),同时模型3d组和1d组也没有差异,这可能和神经元的迟发性死亡有关,电针组内2h和1d组及2h和3d组比较,均存在差异,有统计学意义(P＜0.05),说明早期针刺干预,能够降低脑缺血后神经元的损伤。二、大鼠Y迷宫学习功能检测假手术组大鼠受到电刺激后从非安全区跑至安全区所用时间在各个时间段所用时间均为最少,分别和其他各组(除电针组3d组外)比较差异均有显著性意义(P＜0.01)。模型组之间两两比较没有差异,无统计学意义(P＞0.05),而电针组之间,1d组和2h组存在差异,有统计学意义(P＜0.05),3d组和2h组存在显著差异,有统计学意义(P＜0.01),但1d组和3d组之间没有差异,无统计学意义(P＞0.05)。电针组与模型组比较,电针1d组和3d组均与模型组存在显著性差异,有统计学意义(P＜0.01),但电针2h组与模型2h组没有差异,无统计学意义(P＞0.05)。三、大鼠海马P-ERK1/2的表达假手术组无p-ERK阳性细胞表达,CA1区和CA3区IOD值(SUM)在2h最高,之后就逐渐降低,均有下降趋势。CA1区和CA3区电针各组IOD值(SUM)表达均比模型各组表达低,CA1区和CA3区电针组2h组、1d组、3d组和模型2h组、1d组、3d组比较也有差异,有统计学意义(P＜0.05),CA1区和CA3区电针2h组IOD值(SUM)表达较3d组高,比较均有差异,有统计学意义(P＜0.01)。四、大鼠海马NMDAR1的表达CA1区和CA3区电针各组IOD值(SUM)比模型组均要低,2h最高,之后下降,均有下降趋势。CA1区和CA3区电针2h组、1d组、3d组与模型2h组、1d组、3d组比较有差异,存在统计学意义(P＜0.05)。CA1区和CA3区电针2h组分别和电针3d组比较,都存在显著差异,有统计学意义(P＜0.01)。CA3区模型组2h组和电针2h组存在显著性差异,有统计学意义(P＜0.01)。五、大鼠海马AMPA受体(GluR1)的表达CA1区和CA3区电针各组IOD值(SUM)比模型组均要低,2h最高,且均有下降趋势。电针2h组、1d组、3d组与模型2h组、1d组、3d组比较有差异,也存在统计学意义(P＜0.05)。CA3区电针2h组与模型组2h组比较有明显差异,有统计学意义(P＜0.01),CA1区电针1d组与模型组1d组比较有明显差异,有统计学意义(P＜0.01)。CA1区和CA3区电针2h组和3d组比较,存在显著差异,有统计学意义(P＜0.01)。结论通过神经功能评分的观察说明本次实验造模是成功的,且提示电针对脑缺血大鼠的神经缺损症状有明显改善促进作用。本研究通过电针干预MAPK/ERK信号通路的表达,来观察MAPK/ERK信号通路脑缺血中的作用机制和作用途径,以及与脑缺血损伤和LTP产生和维持密切相关的NMDA受体和AMPA受体的表达,从而阐述MAPK/ERK信号通路与脑缺血间的关系,并对电针干预其机制作相关探讨。本研究发现脑缺血后2h,CA3区ERK磷酸化的表达显著升高,而1d和3d的表达则连续降低,CA1表达比CA3区弱,这可能与2个区对于缺血的耐受不同引起的。且NMDA受体和AMPA受体与ERK磷酸化的表达相一致,这说明MAPK/ERK信号通路在脑缺血中的表达可能与NMDA受体和AMPA受体的表达有关,但在海马各区表达却不尽相同。电针干预后,各指标均比模型组降低,提示:1.电针可以调控MAPK/ERK信号通路,干预脑缺血早期MAPK/ERK信号通路对神经元可能产生的毒性作用,对大脑起到保护作用。2.探讨了MAPK/ERK信号通路脑缺血中的机制,可能主要是通过MAPK/ERK信号通路的激活使NMDA和AMPA等兴奋性氨基酸更易介导,从而对神经元产生毒性作用。3.NMDA和AMPA等受体是LTP产生和维持的重要递质,脑缺血早期MAPK/ERK信号通路的激活和NMDA和AMPA受体的高表达可能对后期存活的神经元的突触重建产生重要影响。本课题创新点:1.在脑缺血模型大鼠中首次系统的观察了针刺对MAPK/ERK的调节干预作用,为临床治疗脑缺血疾病提供了新的理论依据。2.观察了与突触可塑性密切相关的NMDA和AMPA受体的表达,提出了在脑缺血急性期它们的高表达除了产生神经元毒性作用外,可能还会产生病理性的突触可塑性作用,同时电针可以抑制这种作用,为突触重建的早期机制提供了新的思路。更多还原

【Abstract】 Cerebrovascular disease is one of the major diseases,which is a serious hazard to human health.In our country,cerebrovascular disease rank the first three leading causes of death.Ischemic cerebrovascular disease are frequently-occurring and common diseases of mankind,So to find effective treatment,and to explainits mechanism is still the current research in the field of brain science hotspots.In recent years,the study of cerebrovascular disease found that at different stages of post-stroke rehabilitation,the organizational structure and functions of the brain can occur a certain capacity of self-repair and reconstruction.After cerebral ischemia in a few minutes to several months,the cerebral cortex have Significant changes in function and structure,and this change which is called plasticity of the brain occurred not only around in the lesion,but occurred in remote areas.Ischemic brain damage is the most common nerve injury type,and the synapse is the site of transmission and processing of information between the neurons,the function and structure of synapse have significant changes in plasticity with the external stimuli or environmental factors,therefore the synapse is the key part of activity-dependent plasticity changes in brain structure. Extracellular signal regulated kinase,a type of Mitogen activated protein kinases,is a very conservative signal systems that exist in a variety of cells from yeast tomammals.At present,it is more clearly for the research of ERK, and ERK regulate cell proliferation,differentiation and survival.on the one hand to accept from a large number of growth factor,mitogen,environmental stimulation signal,on the other hand,ERK acting on the nuclear transcription factor,regulating gene expression.At present,the search has begun for the ERK pathway in various aspects of the regulatory mechanism for the regulation of signal transduction of the way so as to achieve the purpose of treating disease.Objective To find out the related mechanism,characters,factors and rules between MAPK/ERK pathway and neuronal damage after cerebral ischemia,and to find the effect of electro-acupuncture intervention,provide new foundation to the research of treating cerebrovascular disease by acupuncture and improving the plasticity of brain function.Methods 90 SPF-grade male SD rats were selected and by the drawing method were randomly divided into sham operation group,model group and electro-acupuncture group.Sham-operated group,model group and EA group,each group was divided into 2h(hours),1d(day),3d(days) group,three time period, and a total of 9 groups,10 rats per group.We simulated clinical cerebral syndromes,selected the middle cerebral artery occlusion rat model,used the clinical commonly used electro-acupuncture by acupuncture and electrical stimulation combined,which had determined the effect on cerebral ischemia and sequelae of cerebral ischemia,and treated the focal cerebral ischemia.For the key question of the role of channels and mechanim of ERK pathway in cerebral ischemia,from the multi-link,multi-level,multi-target,we observed the response to electro-acupuncture on ERK pathway and the promoted the role of electro-acupuncture on protective effect of hippocampal neurons in cerebral ischemia.Using "Baihui" and "Dazhui" two points,from the start synapses in hippocampal neurons in area CA1 and CA3,by the way of immunohistochemical and immunofluorescence,we observed cerebral ischemia and the expression after using electro-acupuncture of dynamic changes of ERK1/2,NMDAR1,and AMPA(GluR1) receptors,which is closely related to synaptic plasticity and through the Y maze to testing the learning and memory of rats and the score on the neurological behavior of rats,we had a objective evaluation on models and the results of acupuncture intervention.Thus we deeply reserched the mechanism of acupuncture to promote the protection of neurons of the brain,the role of channels and mechanim between the MAPK/ERK pathway and in early cerebral ischemia the possible mechanism of synaptic plasticity in order to further clarified the plasticity in the brain mechanisms of neurobiology,and provided more exact theory for clinical more good improvement in brain function after cerebral ischemia.Results 1.Neurological signs scoreThe data is analyzed by Spss13.0 statistical,and homogeneity of variance test is P＞0.05,so we use LSD and the S-N-K test to analyze.Sham-operated rats neurobehavioral performance are normal,score of 0 points.Neurological rats showed that the model group,there is no comparison between the two groups statistically significant(P＞0.05),and the acupuncture group 1d and 3d group also have no statistically significant(P＞0.05),however,electroacupuncture group 1d and 3d,respectively,compare with electroacupuncture group 2h are significantly different(P＜0.01).Electroacupuncture 1d group and model 1d group(P＜0.05),electroacupuncture 3d group and model 3d group are between(P＜0.01) differences in comparison.2.Detection of Y maze learning ability in ratSham-operated rats by electrical stimulation of non-security zone from running to a safe area to spend some time in various time periods are used in at least,having significant respectively compared with another groups(apart from electro-acupuncture group 3d)(P＜0.01).the model group is no comparison between the two groups statistically significant(P＞0.05),however between electro-acupuncture groups,1d group compare 2h group with difference(P＜0.05),3d group compare 3h group with difference(P＜0.01),but 1d group compare 3d group with no difference(P＞0.05).Between electro-acupuncture groups and model groups, lectroacupuncture 1d group and 3d group all have statistically significant with model groups,but electro-acupuncture 2h group compare model 2h group with no difference(P＞0.05).3.Hippocampal P-ERK1/2 expression in ratSham-operated group is without p-ERK1/2 positive cells.IOD expression of CA1 area and CA3 area of the electro-acupuncture groups are lower than the expression of model groups,the IOD expression of electro-acupuncture 2h group and modle group 2h are higher than group 1d and group 3d,compared with statistically significant(P＜0.05).electro-acupuncture 2h group,1d group,3d group compare model 2h group,1d group,3d group with difference(P＜0.05). electro-acupuncture groups and model-groups have differences in the corresponding group(P＜0.05).That shows electro-acupuncture at an early stage can reduce p-ERK1/2 expression,which may be the role of neuronal protection.4.Hippocampal NMDAR1 expression in rat IOD expression of electro-acupuncture groups are lower than model groups,and all And have a downward trend,electro-acupuncture 2h group,1d group,3d group compare model 2h group,1d group,3d group with difference(P＜0.05) electro-acupuncture 2h group compare electro-acupuncture 1d group,3d group with difference(P＜0.01).model 2h group and model 3d group compare with difference(P＜0.01).This illustrates that electro-acupuncture at an early stage can decrease the expression of NMPAR1.IOD(SUM) expression of electro-acupuncture groups and model-groups have differences in the corresponding group(P＜0.05).5.Hippocampal AMPA(GluR1)expression in ratIOD expression of electro-acupuncture groups are lower than model groups,and all And have a downward trend,electro-acupuncture 2h group compare model 2h group with differences(P＜0.01).electro-acupuncture 1d group,3d group compare model 1d group,3d group with differences(P＜0.05) electro-acupuncture 2h group compare electro-acupuncture 1d group,3d group with difference(P＜0.01).model 2h group and model 3d group compare with difference(P＜0.01) This illustrates that electro-acupuncture at an early stage can decrease the expression of GluR1.electro-acupuncture groups and model-groups have differences in the corresponding group(P＜0.05).conclusionsThrough the observation of neurological function score that this experimental model was successful,this illustrates that Electroacupuncture can promote the role of significant improvement,on cerebral ischemia in rats with symptoms of nerve defect.At present,MAPK/ERK signaling pathway in the mechanism of cerebral ischemic injury study more,but after cerebral ischemia on synaptic plasticity rarely reported.In this study,through the expression of MAPK/ERK signaling pathway by electroacupuncture intervention,we observe the role of channels and mechanim of ERK pathway in cerebral ischemia and the expression of NMDA receptors and the AMPA receptors closely related toCerebral ischemic injury,the neuro transmitter,which are LTP,an important form of synaptic plasticity,required in the generation and maintenance,thus we can expound the relationship between the MAPK/ERK signaling pathway and cerebral ischemia,and explore its relevant mechanism by electro-acupuncture interventions.This study found that two hours after cerebral ischemia,CA3 area of the expression of phosphorylated ERK was significantly increased,but the expression of one-day group and three-day group continuously reduce,and CA1 than CA3 area is weak expression,this may be related to two areas of different tolerance to ischemia-induced,and the expression of NMDA receptor, AMPA receptor and phosphorylated ERK are in line,this shows that MAPK/ERK signaling pathway in the expression of cerebral ischemia may be related to the expression of NMDA receptor and AMPA receptor,However,the expression them in the hippocampus but not the same district.After electro-acupuncture interventions,all indexes lower than those inmodelgroup,this prompted:1. Electro-acupuncture can control and interfere with MAPK/ERK signaling pathway in the early cerebral ischemia,which may have be neuronal toxicity,and play a role of protecting the brain 2.We have examined the MAPK/ERK signaling pathway in the mechanism of cerebral ischemia,which May be mainly through the activation of MAPK/ERK signaling pathway,make it easier for NMDA and AMPA excited,thus have a toxic effect on neurons.3.NMDA and AMPA receptors is the important neurotransmit of the generation and maintenance of LTP,the activation of MAPK/ERK signaling pathway in the early Cerebral ischemia and the high expression in NMDA and AMPA receptor may be have an important impact on the reconstruction of the synaptic of the late survival of the neurons.Innovation in this topic:1.In Cerebral ischemia model rats for the first time we systematic observated the effect of acupuncture intervention in the MAPK/ERK pathway,and for clinical treatment of cerebral ischemia disease provided a new theoretical basis.2.we observed the expression of the NMDA and AMPA receptors closely related to synaptic plasticity,in the acute phase of the cerebral ischemia,their high expression in addition to the toxic effects on generated neurons,may also have a pathological synaptic plasticity,at the same time EA can inhibit this effect,and for the mechanism of the early synaptic reconstruction provides a new way of thinking.更多还原