【作者】 王咏龙；

【导师】 罗勇；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 博士

【摘要】 背景缺血性脑卒中是神经系统的常见病和多发病,在脑卒中约占75%。大多数患者中遗留有瘫痪、失语等严重后遗症,给患者带来极大的痛苦,同时也给社会和家庭带来沉重的负担。脑缺血/再灌注损伤使脑组织的功能障碍和结构损伤更加严重,使治疗效果大大降低。因此如何采取合理的措施预防和治疗缺血性脑血管病,恢复受损神经细胞功能,降低致残率和病死率,近年来已成为医学界关注的热点课题。脑缺血/再灌注损伤是一复杂的病理生理过程,神经元结构破坏和缺失可能是引起功能障碍的病理关键。目前还没有理想的治疗脑缺血/再灌注损伤的方法。研究发现电刺激小脑顶核对神经系统疾病有神经保护作用,可以抗炎症、抑制细胞凋亡、促进神经组织的结构重建等作用。目前,采用仿生物电,模拟实验性小脑顶核电刺激而研制的小脑电刺激仪已广泛应用于许多神经系统疾病的临床治疗,对多种中枢神经系统疾病都产生了比较肯定的临床效果。Notch1是控制细胞命运的信号传导途径。当Notch1被激活后,神经干细胞进行增殖,而当Notch1活性被抑制时,干细胞则进入分化程序。bHLH基因转录调控因子(包括Hes5和Mash1)是指由一个碱性螺旋-环-螺旋结构及其上游富含碱性氨基酸序列的核酸顺序组成的一类基因,可以调控许多基因的转录。研究表明,Hes5信号通路能抑制神经干细胞向神经元方向分化,促进神经干细胞增殖。Mash1的高表达可促进干细胞的分化,相反低表达则抑制干细胞的分化,使干细胞处于持续的增殖状态。核转录因子-κB对神经干细胞增殖分化作用机制十分复杂,通过对众多涉及炎症和免疫反应等方面的基因调控来影响神经干细胞增殖分化。基于以往实验研究与临床的成果,本实验从电刺激小脑顶核干预局灶脑缺血/再灌注损伤内源性神经干细胞的研究入手,进一步探讨电刺激小脑顶核对大鼠局灶脑缺血/再灌注损伤脑组织神经干细胞的增殖分化的影响及其作用机制,为缺血性脑血管病的治疗提供实验基础。目的1.观察电刺激小脑顶核对大鼠局灶脑缺血/再灌注神经行为学、神经功能缺损的影响以及脑组织的含水量、脑梗死体积的变化。2.观察局灶脑缺血/再灌注各时间点大鼠成体神经干细胞的增殖分化及电刺激小脑顶核对局灶脑缺血/再灌注大鼠成体神经干细胞的增殖分化的影响,探讨电刺激小脑顶对局灶脑缺血/再灌注后神经重建的作用。3.观察电刺激小脑顶核对大鼠局灶脑缺血/再灌注大鼠大脑缺血侧皮质Notchl、Hes5、Mash1和NF-кB p65 mRNA和蛋白表达的变化,进一步探讨电刺激小脑顶核对大鼠局灶脑缺血/再灌注后脑内成体神经干细胞增殖分化影响的作用机制。方法1.改良线栓法制备大鼠大脑中动脉局灶脑缺血1.5h再灌注模型。成年SD大鼠随机分为正常对照组(N组),假手术组(S组),缺血/再灌注组(I/R组),缺血/再灌注后小脑顶核假刺激组(SF组),缺血/再灌注后小脑顶核刺激(F)组。根据再灌注时间的不同又分为ld、3d、7d 3个亚组。对大鼠进行神经功能缺损评分以及测量脑组织的含水量、脑梗死体积的变化。2.制备大脑中动脉局灶脑缺血1.5h再灌注模型。成年SD大鼠随机分为正常对照组(N组),假手术组(S组),缺血/再灌注组(I/R组),缺血/再灌注后小脑顶核刺激(F)组。根据再灌注时间的不同又分为3d、7d、14d、28d 4个亚组,每个亚组动物数n=7只。免疫荧光单标和双标法检测研究局灶脑缺血/再灌注及电刺激小脑顶核对缺血/再灌注大鼠神经干细胞的增殖的动态变化和分化的结果。3.改良线栓法制备大脑中动脉局灶脑缺血1.5h再灌注模型。成年SD大鼠随机分为假手术组(S组),缺血/再灌注组(I/R组),缺血/再灌注后小脑顶核刺激(F)组。根据再灌注时间的不同又分为14d、28d 2个亚组,每个亚组动物数n=7只。采用RT-PCR和Western blotting检测局灶脑缺血/再灌注及电刺激小脑顶核对大鼠局灶脑缺血/再灌注后Notch1、Hes5和Mash1 mRNA及蛋白的表达,采用RT-PCR和免疫组化技术检测NF-кB p65 mRNA及蛋白的表达。结果1.电刺激小脑顶核能提高局灶脑缺血/再灌注大鼠的神经功能缺损评分,降低缺血侧脑组织中脑含水量,缩小脑梗死体积。2.正常对照组和假手术组侧脑室区和缺血周围皮质只存在少量Brdu阳性细胞,局灶脑缺血/再灌注脑损伤后3d大鼠侧脑室下区Brdu阳性细胞显著增加(P<0.01),7d时增加达到顶锋(P<0.01),14d和28d时后Brdu阳性细胞仍增加(P<0.01);局灶脑缺血/再灌注脑损伤后3d大鼠缺血周围皮质Brdu阳性细胞也显著增加(P<0.001),7d时增加达到顶峰(P<0.001),14d和28d时后Brdu阳性细胞仍显著增加(P<0.001);电刺激小脑顶核后大鼠侧脑室下区和缺血周围皮质Brdu阳性细胞增加更明显,与缺血/再灌注组比较,在3d、7d、14d和28d四个时间点Brdu阳性细胞增加更多,均有显著性差异(P<0.05)。免疫荧光双标法显示在正常组、假手术组仅见极少量Brdu/GFAP和Brdu/ DCX双阳性细胞,缺血/再灌注组可见少量Brdu/GFAP和Brdu/DCX双阳性细胞,小脑顶核刺激组可见大量Brdu/GFAP和Brdu/DCX双阳性细胞,Brdu/GFAP和Brdu/DCX双阳性细胞明显增多,主要分化为神经元和神经胶质细胞。3.与假手术组比较,脑缺血/再灌注组和脑缺血/再灌注后小脑顶核刺激组Notchl、Hes5、Mash1 mRNA和蛋白的表达量均增加(P<0. 01);与脑缺血/再灌注组比较,小脑顶核刺激组Notchl、Hes5、Mash1 mRNA和蛋白的表达量增加更明显(P<0.05)。与假手术组比较,脑缺血/再灌注组和脑缺血/再灌注后小脑顶核刺激组NF-кB p65 mRNA和蛋白的表达量均增加(P<0.01);与脑缺血/再灌注组比较,小脑顶核刺激组NF-кB p65 mRNA和蛋白的表达量明显降低(P<0. 05)。结论1.成功建立改良线栓法大鼠局灶脑缺血/再灌注模型。电刺激小脑顶核可以降低脑缺血/再灌注损伤后急性期死亡率,促进肢体功能恢复。电刺激小脑顶核能提高大脑中动脉局灶脑缺血/再灌注模型大鼠的神经功能缺损评分,降低缺血侧脑组织中脑含水量,缩小脑梗死体积,从而达到脑保护作用2.脑缺血后自身的神经干细胞也存在增殖。在正常对照组和假手术组大鼠SVZ和缺血侧皮质有少量神经干细胞,未见神经干细胞增殖。内源性神经干细胞增殖在脑缺血再灌注后逐渐升高,在脑梗死后7d达到高峰,以后增殖逐渐下降,28d神经干细胞仍有增殖。电刺激小脑顶核后神经干细胞增殖更明显,在7d达到高峰,28d后在SVZ和缺血周围皮质神经干细胞均还有明显增殖。电刺激小脑顶核能促进脑缺血后大鼠内源性神经干细胞的增殖。在正常组、假手术组仅见极少量神经干细胞分化,缺血/再灌注组可见少量神经干细胞分化,小脑顶核刺激组可见大量神经干细胞分化,分化明显增多,分化为神经胶质细胞和神经元。电刺激小脑顶核促进神经干细胞分化为神经胶质样细胞和神经元样细胞。电刺激小脑顶核通过促进内源性神经干细胞增殖和分化发挥脑组织神经重建作用。3.正常成年大鼠脑组织Notchl、Hes5、Mash1和NF-кB p65表达较少。局灶脑缺血/再灌注后,大鼠缺血侧脑皮质Notchl、Hes5、Mash1和NF-кB p65 mRNA及蛋白表达明显增强,电刺激小脑顶核后Notchl、Mash1和Hes5 mRNA及蛋白表达增强更明显,NF-кB p65 mRNA及蛋白表达增强则减弱。电刺激小脑顶核通过促进Notchl、Hes5和Mash1 mRNA及蛋白表达和抑制NF-кB p65 mRNA及蛋白表达来影响神经干细胞增殖和分化。更多还原

【Abstract】 BackgroudIschemic stroke is the disease of nervous system,which is commonly seen and frequently occurs. Among all the types of stroke,ischemic stroke occupies the stroke disease above 75%. Most of the stroke patients have some scrious sequelas,such as paralysis,aphasia,and et al. It not only torments the stroke patients,but also bring the society and the patients, families heavy burden. Cerebral ischemia/reperfusion cause the cerebral functional impairment and structure damage become worse,which degrades the therapeutic efficacy obviously.The appropriate methods of prevention and therapy for ischemia/reperfusion to restore the function of damaged neurons and to reduce disability and mortality remain a subject of hottest focuses in recent years. Cerebral ischemia/reperfusion has complicated pathophisiological process and the specific mechanism. Functional impairment could be caused neuron disorganization and depletion which performed important pathological significance in cerebral ischemicin jury. At present,there is no satisfactal treatment of cerebral ischemia/reperfusion jury in study and clinical application. Studies showed that FNS has exact neuroprotective effects against damage of nervous system diseases, include anti-inflammatory,supress cell apoptosis and promoting nerve tissue re- construction. Cerebellum electric stimulation devices which start from experimental fastigial nucleus stimulation(FNS) and minic biological electric current have applied in clinic for treating some disease and showed the satisfied clinical effects on several central nervous system diseases recently. The signal pathway of Notch1 can control destiny of the cell. The neural stem cell carries on proliferation after Notch1 is activated and the neural stem cell enters the procedure of differentiation when the activeness of Notch1 is suppressed. The basic helix-loop-helix factor of bHLH gene richly contains an sequence of alkalinity amino acid in its upstream and regulate transcription of many genes. The research indicates that the Hes5 signal passage can inhibit differentiation of neural stem cell to the neuron and promote proliferation of neural stem cell. Mash1 may promote the differentiation of neural stem cell when the expression of Mash1 is high and cause condition of proliferation through the suppression of differentiation of nerve stem cell when the expression of Mash1 is low. There is a very complex mechanism of NF-κB in regulation proliferation and differentiation of neural stem cell which involves the aspect of inflammation and immune response. Based on the past clinical and experimental achievements, this project is to proceed with how FNS influence the endo- genesis neural stem cell and research the proliferation and differentiation of endogenous neural stem cell and mechanism with FNS. With the project carried out,we can get the experimental basis for the clinical treatment of ischemic stroke.Objectives(1) The protection of FNS was observed from the aspects of neural function comprehend evaluation, brain tissue water content and cerebral infarction size.(2) To identify proliferation and differentiation of neural stem cells in the adult rats which were treated with FNS at each time point of ischemia/reperfusion respectively and to investigate the possible effect of FNS on the plasticity of nerve of the brain.(3)The change of mRNA and protein expression of Notch1, Hes5, Mash1 and NF-кB p65 in each group was assessed to study on the mechanism of proliferation and differentiation of neural stem cells in focal cerebral ischemia/reperfusion rats.Methods(1) Focal cerebral ischemia/reperfusion injury was induced by intraluminal middle cerebral artery occlusion (MCAO) method using a monofilament thread in rats and and treated with FNS after the reperfusion was performed. Adult SD rats were randomly devided into five groups: normal control group(N group),sham-operation control grop(S group),ischemia/reperfusion group(I/R group),ischemia/ reperfusion treated with FN sham-stimulation group(SF group) and ischemia/reperfusion treated with FNS group(F group). Each group contained three time points of 1d,3d and 7d. The protective effect of FNS was observed from the aspects of neural function comprehend evaluation, brain tissue water content and cerebral infarction size.(2) Animal model was made by ligating external carotidartery and inserting a piece of nylon thread into the internal carotidarter. Adult SD rats were randomly devided into four groups: normal control group(N group),sham-operation control group(S group),ischemia/reperfusion group(I/R group)and ischemia/reperfusion treated with FNS group(F group). Each group contained four time points of 3d,7d,14d and 28d . There were seven rats in each group at each time point. Single and double immunofluorescence histochemical method was used to identify proliferation and differentiation of neural stem cells in the adult rats which were treated with FNS after ischemia/reperfusion at each time point.(3) Focal cerebral ischemia/reperfusion injury was induced by MCAO method. Adult SD rats were randomly devided into three groups: sham-operation control group(S group),ischemia/reperfusion group(I/R group) and ischemia/reperfusion treated with FNS group(F group). Each group contained two time points of 14d and 28d. There were seven rats in each group at each time point. The mRNA and protein expression of Notch1,Hes5 and Mash1 were assessed by western blotting and RT-PCR and the mRNA and protein expression of NF-кB p65 was assessed by RT-PCR and immunohistochemistry stain.Results(1) The results showed that FNS can increase scores of comprehensive neurological function and decrease brain water content and reduce the volume of infarct volume.(2) Only a small amount of Brdu-labeled cells was found in SVZ and in cortex in the result of immunofluorescence histochemical method in normal control group and sham-operation control group. After three day of focal cerebral ischemia/reperfusion ,t he number of Brdu-labeled cells in SVZ increase significantly(P<0.01) and increased to a peak after seven days of focal cerebral ischemia/reperfusion(P<0.01), then the rate of increase descended after fourteen days and twenty eight days of focal cerebral ischemia/reperfusion(P<0.01). After three day of focal cerebral ischemia/reperfusion, the number of Brdu-labeled cells in cortex increase significantly(P<0.001) and increased to a peak after seven days of focal cerebral ischemia/reperfusion(P<0.001), then the rate of increase descended after fourteen days and twenty eight days of focal cerebral ischemia/reperfusion(P<0.001). Compared with group of focal cerebral ischemia/reperfusion at the time point of three days,seven days,fourteen days and twenty eight days,the number of Brdu-labeled cells in SVZ and in cortex increased more strikingly in the group of focal cerebral ischemia/reperfusion treated with FNS , the difference was significant(P<0.05). There were a very small amount Brdu/DGX- labeled and Brdu/GFAP-labeled cells which were found in normal control group and sham-operation control group and only a small amount of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found in focal cerebral ischemia/reperfusion group. A large amount of number of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found in F group. The proliferated neural stems cells were induced to differentiate into glia cells and neurons.(3)Compared with sham operation control group, the mRNA and protein expression of Notch1 , Hes5,Mash1 and NF-кB p65 increased in ischemia/reperfusion group and ischemia/reperfusion treated with FNS group(P<0.01). Compared with ischemia/reperfusion group, the mRNA and protein expression of Notch1,Hes5 and Mash1 increased and the mRNA and protein expression of NF-кB p65 decreased in ischemia/ reperfusion treated with FNS group(P<0.05).Conclusions(1)Focal cerebral ischemia/reperfusion injury model was established successfully. The application of FNS could lower the mortality of rats in the acute stage of ischemia/reperfusion injury.The application of FNS could increase scores of comprehensive neurological function,decrease brain water content and reduce the volume of infarct volume. FNS can protect brain after ischemia/reperfusion injury.(2) There was proliferation of neural stem cells in SVZ and cortex after ischemia/reperfusion injury. Only a small amount of Brdu-labeled cells were found in SVZ and in cortex in adult rats. The proliferation of endogenous neural stem cells in SVZ and cortex gradually increased after ischemia/reperfusion injury,the rate of increase gradually slowed down after the peak of 7d time point. Proliferation of neural stem cells is more obvious after focal cerebral ischemia/reperfusion treated FNS than only focal cerebral ischemia/reperfusion,the rate of increase gradually slowed down after the peak of 7d time point. There were a very small amount Brdu/DGX-labeled and Brdu/GFAP-labeled cells which were found in normal control group and sham-operation control group and only a small amount of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found in focal cerebral ischemia/reperfusion group. A large number of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found after focal cerebral ischemia/reperfusion treated FNS. The proliferated neural stems cells were induced to differentiate into neurons and glia cells. FNS could promote the differentiation of neural stem cells to into glial-like cells and neuron-like cells. FNS plays a role in nerve reconstruction of brain tissue by promoting the proliferation and differentiation of neural stem cells.(3) The mRNA and protein expression of Notch1,Hes5,Mash1 and NF-кB p65 was low in adult rats and the mRNA and protein expression of Notch1,Hes5 ,Mash1 and NF-кB p65 increase after ischemia/ reperfusion and ischemia/reperfusion treated with FNS. Compared with only ischemia/reperfusion,there was a significant increase of the mRNA and protein expression of Notch1,Hes5 and Mash1 and a significant de- crease of the mRNA and protein expression of NF-кB p65 after ischemia/ reperfusion treated with FNS. FNS could influence proliferation and differentiation of neural stem cells by promoting the mRNA and protein expression of Notch1,Hes5 and Mash1 and inhibiting mRNA and protein expression of NF-кB p65.更多还原