【作者】 李源莉；

【导师】 陈建宗；

【作者基本信息】 第四军医大学 ， 中西医结合临床， 2007， 硕士

【摘要】 脑血管疾病(Cerebrovascular disease, CVD)是各种血管源性脑病变引起的脑功能障碍,是神经系统的常见病和多发病,死亡率约占所有疾病的10%,是目前人类疾病三大死亡原因之一,50%～70%的存活者遗留瘫痪,痴呆及失语等严重残疾,给社会和家庭带来沉重负担。而缺血性脑血管病是脑血管病中最常见的一种,约占脑血管病70～80%。痴呆是CVD后期的常见并发症,有研究报告发现,78%的脑血管病人伴有不同程度的认知能力下降,痴呆也是老年人致残的常见原因,在65～70岁老年人发病率为1.4%,85岁以上达到23.6%。痴呆的典型病理变化为大脑皮质、海马等与认知记忆有关部位的神经元大量变性死亡,数目明显减少。目前脑血管疾病发病率呈逐年上升趋势,因此防治CVD后期所引起的神经细胞损伤方面的研究具有重要的现实意义。以往研究发现大脑短暂性缺血可导致皮质海马部位神经元大量变性凋亡,从而引起了患者认知功能障碍。近年来研究发现线粒体功能障碍在神经细胞凋亡过程中具有关键性作用,线粒体是细胞内的能量工厂,细胞缺血缺氧时可引起线粒体氧化呼吸链电子传递障碍,线粒体内部细胞凋亡信号转导通路启动。Caspase凋亡信号通路是目前认为比较经典的路径,线粒体功能障碍时可激活Caspase家族蛋白,首先活化的Caspase-9,依次活化下游蛋白,最终导致细胞不可逆死亡。因此探索抑制Caspase相关家族蛋白活化,对于防止细胞凋亡具有重要意义。人参皂甙Rg1(Ginsenoside Rg1)是人参重要的活性成分之一,近年来人参皂甙Rg1的的神经营养和神经保护作用受到人们的关注,研究表明其对帕金森病(parkinson,s disease,PD)发病过程中黑质多巴胺能神经元变性凋亡具有一定的抑制作用,提示其可能在防治PD病变中具有积极作用,然而人参皂甙Rg1对于大脑皮质神经细胞损伤是否也具有保护作用,值得进一步研究。NKs是一个结构相似的速激肽家族,主要包括P物质(Neurokinin-1,NK1)、神经激肽A(NK2)、神经激肽B(NK3),其生物学效应由相应的NK1、NK2和NK3受体(G蛋白偶联受体)来介导。研究表明神经激肽-神经激肽受体在中枢神经系统分布十分丰富,提示神经激肽在神经元生理和病理过程中可能具有重要作用。NK-4R是研究发现的一种新型神经激肽受体,目前对于其在中枢神经系统的分布特征还不清楚。因此本课题的主要目的在于以前几个方面:(1)观察NK-4R在大脑皮质部位神经细胞上是否有分布以及分布特征;(2)观察人参皂甙Rg1是否对缺血再灌注致皮质神经元损伤具有保护作用;(3)探讨人参皂甙Rg1神经保护作用机制是否与抑制Caspase家族蛋白活化有关。这些问题的研究将为脑血管疾病后期神经细胞损伤凋亡以及痴呆的防治提供一些实验依据。本课题所采用的实验方法包括:采用动物实验研究、短暂反复结扎小鼠双侧颈总动脉制备脑缺血再灌注损伤模型、给予人参皂甙Rg1进行药物干预、采用免疫细胞化学染色,荧光双标及激光共聚焦显微镜技术等方法,主要探讨和分析NK-4R在大脑皮质的分布特征;NK-4R标记的大脑皮质神经元在人参皂甙Rg1药物干预组,模型损伤组以及空白对照组之间是否存在数目变化差异;人参皂甙Rg1的神经保护机制是否与粒体功能障碍时Caspase家族蛋白活化有关。主要实验结果包括:(1) NK-4R免疫阳性产物在大脑皮质的锥体细胞层(Ⅲ、Ⅵ层)分布最多,在皮质内、外颗粒层、多形层(Ⅱ、Ⅴ、Ⅶ层)也有分布,其阳性产物表达于神经元胞体和突起的膜表面,是一种细胞膜受体。免疫荧光双标结果观察到NK-4R与神经元胞核特异性标记物NeuN共存(100%,146/146,双标细胞数目/NK-4R阳性细胞数目),且不与星形胶质细胞标记物GFAP共存,证实NK-4R确实是分布于神经细胞膜上。(2)人参皂甙Rg1对小鼠脑缺血再灌注致皮质神经元损伤存在一定的神经保护作用。人参皂甙Rg1能够增加神经细胞活力,减少皮质大脑皮质神经元的死亡数目。而模型组小鼠神经细胞活力降低,存活的神经元的死亡数目明显少于给药组。(3)人参皂甙Rg1神经保护机制可能与Caspase凋亡通路中一个非常重要的因子,Caspase-9活性的抑制有关。模型组动物大脑皮质神经细胞核Caspase-9表达数目增加,显著多于空白组和人参皂甙Rg1中剂量组。主要结论:(1) NK-4R免疫阳性产物在大脑皮质神经元的分布表达,提示NK-4R可能参与了哺乳类动物大脑皮质神经元功能调节和病变过程。(2)人参皂甙Rg1能够抑制大脑皮质神经元缺血导致的损伤凋亡,表明一定剂量的人参皂甙Rg1可能具有阻止脑血管疾病中神经细胞损伤的作用。(3)人参皂甙Rg1神经保护机制可能与降低Caspase-9的表达有关,此机制还有待于进一步探讨。本实验为NK-4R的进一步研究提供了一些形态方面的依据;同时也为人参皂甙Rg1防止脑血管疾病后期的神经病变的防治给出了一些实验参考;神经细胞凋亡是一个由多个因素参与的非常复杂病变过程,我们的实验观察到人参皂甙Rg1可能是通过了抑制caspase-9的表达,此作用机理仍需进一步探讨。更多还原

【Abstract】 Cerebrovascular disease (CVD)is a dysfunction of brain disease induced by many kinds of abnormity in vascular and blood flow. it is a common and frequently-occurring disease in nervous system, which death rate probably rise to 10% and the third cause of death in all diseases. The later complications of CVD lead to 70%～80% survivors remain some severe symptoms, such as paralysis、dementia、aphasia. The ischemic CVD is a common form of CVD, probably up to 70%～80%. Dementia is a progressive disorder which is accompanied by cognitive decline and behavioural problems, which is one of the most common complication of CVD. Dementia could cause of disability in the aged population. It is reported that the 78% patients of CVD showed obstacle in cognitive in different degrees. The typical changes of pathology in Dementia is that the number of neurons in cerebral cortex and hippocampus displayed decreased significantly. the incidence of CVD shows increase every year, the later pathological changes may probably make the patients dementia which put big burden on family and society, therefore it is important to find methods for preventing and remeding apoptosis of neurons in cerebral cortex and hippocampus. Previous studies have indicated that Mitochondrial dysfunction may be one of crucial factors implicating in degeneration of neurons in Dementia, Mitochondrial is an energy factory in cells, when cell occur ischemia, the chain of electron transmission in Mitochondrial develop obstacle, the road of apoptosis signal priming in cell, at last cell death. Caspase pathway is presumed to be a typical rode to apoptosis, the first actived protein is capase-9, then caspase-3/6/7. So Caspase-9 is a key determiner in neuronal death and neurodegeneration. it is necessary, thus, to investigate novel strategies on preventing neurons from ischemia lesions and improving the treatment of dementia.Ginsenoside Rg1 is one of major important active component of Ginsenoside , in recent years, the neuroprotective effect of Ginsenoside Rg1 has arised researchers concern. It is reported that Ginsenoside Rg1 could inhibit apoptosis of dopamine neurons in substantia nigra in PD. It is necessary to research that whether Ginsenoside Rg1 would perform neuroprotective in cell death in cerebral cortex induced ischemia or not , which would provide some experimental datas for further study in apoptosis in CVD and dementia.Mammalian neurokinins [NKs] are a family of tachykinin peptides that include substance P (SP; neurokinin-1, NK-1), neurokinin A (SK; NK-2; neurokinin A) nd neurokinin B (NK; NK-3; neurokinin B). Their biological functions are mediated by three distinct G-protein coupled neurokinin receptors, namely SP receptor (SPR: NK-1 receptor, NK-1R), neurokinin A receptor (NK-2R) and neurokinin B receptor (NK-3R). Neurokinin peptides and neurokinin receptors are abundantly distributed in the CNS and known to significantly interact with neurons. Previous evidence has suggested that they may be involved in the regulation of physiological and pathological processes in neurons.Aims of the experiment including:(1)To examine the localization and distribution patterns of neuronkinin-4 receptor (NK-4R), a new style of NKs receptor, in neurons of the cerebral cortex of adult C57/BL mice;(2)later we used the NK-4R marked the survival neurons in cerebral cortex, Further studies should be devoted to elucidate whether Ginsenoside Rg1 are involved in protecting degenerative death of neurons in cerebral cortex in CVD;(3)To observe the mechanism in neuroprotection of Ginsenoside Rg1 is whether involved in modulating the active of caspase-9.Methods of the experiment is as follows:Roles of Ginsenoside Rg1 in regulation of activity of neurons in cerebral cortex and possible mechanism interaction with caspase pathway were studied by using animal models induced by ischemia leison, administration of Ginsenoside Rg1, then by using Nissl staining, immunoenzyme histochemistry and double immunofluorescent staining, the distribution patterns of NK-4R in neurons in the layers of cerebral cortex, the localizations of NK-4R and neuronal nuclear specific protein (NeuN ), NK-4R and glial fibrillary acidic protein (GFAP) , the expression of caspase-9 in the neurons of cerebral cortex of adult mice were observed, their distributions were analyzed by semi-quantitative method, double immunofluorescence, laser scanning confocal microscopy .Major results are including: (1) NK-4R-immunoreactivity was found extensively localized in neuronal somata and neurite of cerebral cortex (layerⅡ、Ⅳ、Ⅵ) , and mainy in layerⅢ,Ⅴ.Double-labeling experiments confirmed that 100% NK-4R-immunoreactive neurons co-expressed NeuN, but not GFAP; (2) Roles of Ginsenoside Rg1 in regulating ischemia lesion induced neurons death were applied in animal experiment. Pretreatment with Ginsenoside Rg1 increased cell activity and numbers of NK-4R marked the survival neurons in cerebral cortex; (3) Roles of Ginsenoside Rg1 in regulating ischemia lesion were perfomed by in vivo animal models. Comparison with that of single ischemia lesion group, treatment with Ginsenoside Rg1 increased the number of NK-4R-stained survival neurons and decreased the number of caspase-9 expression, while single ischemia lesion group showed decreased the number NK-4R-stained neurons and increased number of caspase-9 expression in neurons of cerebral cortex.We have concluded: (1) The localization and distribution of NK-4R in the neurons of cerebral cortex of mice suggests that the novel NK-4R may be involved in the modulation of neuronal properties and the process of neuronal pathological changes in the cerebral cortex of mammals; (2) Intervention effects of Ginsenoside Rg1 on ischemia lesion of neurons in cerebral cortex have indicated that Ginsenoside Rg1 may play neuroprotective role in CVD and Dementia; (3) we also infered that the mechanism neuroprotective of Ginsenoside Rg1 were probably involved in inhibition the active of caspase-9.Our experiment showed the distribution of NK-4R in cerebral cortex; Taken together with previous studies, the present results have indicated that Ginsenoside Rg1 may be implicated in degeneration of neurons and pathogenesis of CVD and Dementia. Our datas also suggest that Ginsenoside Rg1 may function as inhibition in the active of caspase-9. However, apoptosis is a complicated process that many factors and pathways take part in, it is needed to study the neuroprotective mechanism of Ginsenoside Rg1 involved in caspase-9 further.更多还原