【作者】 于淼；

【导师】 钱涛； 高维娟；

【作者基本信息】 承德医学院 ， 肿瘤学， 2010， 硕士

【摘要】 脑血管疾病是目前人类死亡和残疾的主要原因之一,该病的发生主要与脑血流的减少和缺血性脑损伤有关。脑缺血后的许多继发因素如研究较多的兴奋性氨基酸毒性、细胞内钙超载、梗死灶周围的去极化、炎症反应、一氧化氮、自由基引起的脂质过氧化损伤等,都可诱导神经细胞死亡,从而引起脑损伤。但脑缺血后神经细胞死亡的确切机制目前尚无定论。研究脑缺血后神经元损伤的发生机制以及可能的干预因素,寻找有效的脑保护药物,最大限度地减少神经细胞的急性和迟发性死亡,保护脑功能,仍是当前研究的热点和难点。有研究表明,中枢神经系统的某些神经元对缺血缺氧有选择性易损性,其中对缺血最敏感的是海马神经元CA1区,且发生损害的时间最迟。研究发现在全脑缺血再灌注后12小时海马CA1区神经元无变化,24小时神经元排列整齐、规则,而在缺血再灌注后7天CA1段大部分神经元消失,残存的胞体固缩,胞浆空化。由此可见在脑缺血再灌注损伤中存在迟发性神经元死亡。应用干预措施,减少迟发性神经元死亡有着重要的意义。中药黄芪,应用历史悠久,具有抗炎、抗病毒、抗衰老、调节免疫、抗肿瘤等作用,并有实验表明对缺血后迟发性神经元损伤有保护作用,受到医家的青睐。鉴于中药黄芪对脑血管病多靶点治疗的作用特点,黄芪对缺血性脑损伤影响的研究也成为当前医学领域研究的热点。本实验的研究目的是,应用四血管法建立全脑缺血再灌注损伤模型,观察脑缺血再灌注7天海马CA1区神经元形态学和超微结构变化,存活细胞数目和细胞凋亡率的变化,以及大脑皮质中凋亡相关蛋白P53、Bcl-2表达的变化。并应用黄芪注射液进行干预,对上述指标的变化情况进行比较,探讨黄芪注射液减轻脑缺血再灌注损伤的作用机制,为黄芪注射液的临床应用提供理论基础。实验方法:本实验中我们应用成本低,种系纯合性好,与人类脑血管解剖特性相似的SD大鼠为实验动物,采用改良的Pulsinelli四血管法,建立全脑缺血15分钟再灌注7天损伤模型,并观察黄芪注射液的作用。此法的优点是,检验缺血是否成功的指标明确,能在清醒的动物上复制。我们将48只SD大鼠随机分为假手术组,缺血再灌注模型组,尼莫地平组,黄芪注射液治疗组。并选择应用HE染色在光学显微镜下观察海马CA1区神经元形态变化及存活神经元计数;应用电镜观察海马CA1区超微结构变化;应用流式细胞仪检测海马组织神经元凋亡率;应用生化试剂盒检测脑缺血再灌注7天大脑皮质中P53、Bcl-2的表达。操作严格按说明书进行。检测结果:(1)海马CA1区神经细胞形态学变化:假手术组:海马CA1区神经元外形规则,3～4层,细胞层排列整齐而致密,核大而圆,透明,有1～2个明显核仁,核膜清楚。模型组:神经元排列紊乱而疏松,外形皱缩,部分细胞脱失,核固缩深染,胞浆空化,核溶解,核膜境界不清;神经元计数显著低于假手术组(P<0.01)。尼莫地平组和黄芪注射液治疗组相似:大部分神经元保持正常形态,细胞排列基本规则,少量细胞轻度脱失,部分胞染变深,残存的神经元大部分呈可逆性缺血性改变;神经元计数显著高于模型组(P<0.01)。(2)海马CA1区神经细胞超微结构变化:假手术组:7天后神经元细胞核呈卵圆形,两层膜结构清晰光滑,核周间隙均匀,核内染色质均匀分布,胞浆内细胞器丰富,结构完整,可见高尔基体分布,结构呈扁平囊泡平行排列,线粒体呈圆形、椭圆形,内嵴清晰可见,基质均匀,粗面内质网平行排列或散在分布,大量糖原颗粒存在于胞浆中。模型组:细胞核膜结构模糊,线粒体空泡化明显,且较多较重,高尔基体不规则。尼莫地平组和黄芪注射液治疗组相似,与模型组相比,海马病变较轻,胞浆内细胞器丰富,细胞器结构较完整,部分细胞器结构变性。(3)海马神经元凋亡率:与假手术组相比,模型组神经元凋亡率明显升高(P<0.01);与模型组相比,黄芪注射液组和尼莫地平组神经元凋亡率显著降低(P<0.01);与尼莫地平组相比,黄芪注射液组神经元凋亡率无明显变化(P>0.05)。(4)大脑皮质凋亡相关蛋白P53、Bcl-2表达的变化:假手术组:P53无明显表达,Bcl-2可见表达。与假手术组相比,模型组P53阳性细胞显著增多,Bcl-2阳性细胞明显减少(P<0.01);与模型组相比,尼莫地平组和黄芪注射液治疗组相似,P53阳性细胞明显降低,Bcl-2阳性细胞明显增多(P<0.01)。与尼莫地平组相比,黄芪注射液治疗组P53、Bcl-2阳性细胞无明显差异(P>0.05)。结论:黄芪注射液可明显改善脑缺血再灌注后海马CA1区神经元形态学及超微结构变化,减少神经元脱失,降低凋亡率;降低P53、增高Bcl-2的表达,从而保护脑组织。更多还原

【Abstract】 Cerebrovascular diseases are one of the causes of death and disability in the world,and they are closely related to disturbances of blood flow and ischemia. Recent studies found that ischemia brain damage is resulted from many reasons such as functional rearrangement of glia cellsan ion balance disorder,lipid peroxidation induced by free radicals,and changes of neurotransmitters.However the mechanisms underlying ischemia-induced cell death are currently unclar.The neural cell injury after studying a cerebral ischemia the occurrence mechanism and possible intervention factor of the harm,looking for the valid brain protection medicine,utmost reducing a neural cell of impatient and delayed neuronal death,protecting the brain function,are still a little bit hot and crux to study at present.There is the research enunciation,some neural cells of the central nervous system have selectivity to ischemia and is harmful easily.Among them most impressionable to ischemia is hippocampal CA1 region and take place time of the damage at the latest.The research notes that 12 hours after the global cerebral ischemia-reperfusion,the neurons in the rats hippocampal CA1 region has no variety.24 hours, the neurons pyramidal cells ranged regularly,but in the I/R 7 d,the number of normal neuron decreased,the cell body shrank,and the nucleus became out of shape,characterized by hippocampal structure destroyed,cell swelling,cell lysis,pyknotic nuelei,and vacuolization.So there were existed delayed neuronal death in the cerebral ischemia injury.Applying the intervention measure and reducing delayed neuronal death have important meaning.Chinese herbal medicine astragulas what apply history is long,have anti- inflammation,virussafe,anti-decrepitude, adjust-immunity,anti-tumor etc.And some experiment show a conclusion that astragulas have protection function in the global cerebral ischemia-reperfusion injury,being subjected to favor of cure the house.Combine owing to Chinese herbal medicine astragulas to cerebral disease many parts order the function characteristics of the treatment,To study the protective effects of astragulas against the whole cerebral ischemia and reperfusion injury and its mechanisms,becomes current medical science realm to study of a little bit hot.The research purpose of this experiment: Applications was established by four-vessel cerebral ischemia-reperfusion injury model to observe the cerebral ischemia-reperfusion 7 days,hippocampal CA1 neurons morphological and ultrastructural changes, the number of survival cells and apoptosis rate of change, and Cortex apoptosis-related protein P53, Bcl-2 expression changes.And application of astragalus to intervene, to change these indicators to compare and explore Astragalus reduce cerebral ischemia-reperfusion injury mechanism for Astragalus theoretical basis for clinical applications.Experimental methods:In this experiment we applicate the SD rat whose cost is low and who growing to fasten purely match sex good,its cerebral with mankind of the characteristic likeness to be used as the experiment animal;We adopt Pulsinelli four vessels occlusion for 15 min and reperfusion for 7d in rats.This experiment was performed to evaluate effects of astragulas injection.48 rats were randomly assigned to sham group,I/R model group,nimodipine group and astragulas injection group. And select the application of HE staining were observed under the optical microscope, hippocampal CA1 neurons in morphological changes and survival neuron counts;application of electron microscopy ultrastructure of hippocampal CA1 area;application of flow cytometry in hippocampal neuronal apoptosis rate; biochemical test kit cerebral ischemia in the cerebral cortex 7 days P53, Bcl-2 expression. Operations carried out in strict accordance with instructions.Examination result: (1)The morphologic characteristics of neurons in the rat hippocampal CA1 region:Sham group:In there were 3～4 layers neurons ranged regularly in hippocampal CA1 region,the nuclcus was big,round,and pellucid,which can be seen 1～2 nucleolus clearly.I/R model group: Neurons disordered and loose, wrinkled appearance, some cells drop out, deeply stained nuclear condensation, cytoplasmic cavitation, nuclear dissolution, nuclear realm unclear;neuron counts were significantly lower than the normal control group(P<0.01).Nimodipine group and Astragulas injection group: Most of the neurons to maintain a normal shape, cell arrangement of the basic rules,some intracellular staining, become darker in the majority of the remaining neurons was reversible ischemic changes in neurons counts were significantly higher than the I/R model group(P<0.01). (2) Hippocampal CA1 area ultrastructural changes of nerve cells: Sham group: 7 days after the neuron nucleus was oval, two clear and smooth membrane, perinuclear space uniform, evenly distributed nuclear chromatin, abundant cytoplasmic organelles,structural integrity, we can see the distribution of Golgi structure was parallel flat vesicles, mitochondria were round, oval shape, clearly visible cristae, matrix uniform, rough endoplasmic reticulum arranged in parallel, or scattered in a large number of glycogen particles present in the cell Pulp.I/R model group: nuclear membrane structure fuzzy,mitochondrial vacuolization evident, and the more heavier, irregular Golgi. Nimodipine group and Astragalus injection group compared with the I/R model group, hippocampus lesions lighter cytoplasmic organelles rich organelle structure is more complete, some degeneration of organelle structure. (3)Hippocampal neuronal apoptosis rate: Compared with sham group, neuronal apoptosis rate in I/R model group was obviously increased(P<0.01); Compared with I/R model group, Nimodipine group and Astragalus injection group of neuronal apoptosis rate was significantly reduced (P<0.01). Compared with nimodipine group, Astragalus injection group neuronal apoptosis rate was no significant change(P>0.05).(4) Cortex apoptosis-related protein P53, Bcl-2 expression: Sham group: P53 expression was not changed significantly, Bcl-2 expression of the more obvious. Compared with sham group, I/R Model group P53 expression was significantly increased, Bcl-2 expression was significantly reduced (P<0.01). Compared with I/R Model group,Nimodipine group and Astragalus injection group P53 expression were significantly inhibited, Bcl-2 expression were significantly increased(P<0.01). Compared with nimodipine group, Astragalus injection group P53, Bcl-2 expression was no significant change(P>0.05).Conclusion: Astragalus can significantly improve the cerebral ischemia-reperfusion in hippocampal CA1 neurons after the morphological and ultrastructural changes, reduction of neuronal loss, reduce the hippocampal neuronal apoptosis rate, reduce the expression of P53, increased expression of Bcl-2. Astragalus can protect the brain tissue.更多还原