【作者】 卞干兰；

【导师】 管振龙； 陈良为；

【作者基本信息】 河北师范大学 ， 动物学， 2007， 硕士

【摘要】 帕金森病(Parkinson’s disease, PD)是一种常见的中枢神经变性疾病,患者出现静止性震颤、运动迟缓、僵硬、步态异常和姿势不稳等严重的运动症状。PD的典型病理变化为大量黑质多巴胺神经元变性死亡,现行的PD治疗方法不能阻止黑质神经元进行性死亡和PD病发生。以往研究表明在引起黑质多巴胺神经元死亡和PD发生的多种因素中,谷氨酸兴奋性毒性可能是重要原因之一。因此,探索阻止谷氨酸兴奋性毒性损伤和保护黑质多巴胺神经元的新手段,对于完善PD治疗十分必要。神经激肽(Neurokinins)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-神经激肽受体在基底核分布十分丰富,它们与基底核神经元之间存在显著的相互作用,提示神经激肽在基底核生理和病理发生的过程中可能具有重要作用。我们研究组以往的实验已初步证明神经激肽分子能够干预黑质神经元兴奋性毒性损伤,特别是发现神经激肽NK3受体激动剂能产生兴奋性毒性的协同效应。因此,进一步确认神经激肽NK3受体拮抗剂能否调节黑质多巴胺神经元兴奋性毒性损伤,具有重要价值。本课题采用Fluoro-Jade C(FJC)染色、MPTP(1-methy-4-phenyl-1,2,3,6- tetrahydropyrindine)和KA(Kainic acid,红藻氨酸)损伤动物模型、神经激肽NK3受体特异性拮抗剂干预方法、免疫组织化学染色、动物行为学分析等方法,研究FJC方法在MPTP和KA动物模型中的应用,以及神经激肽NK3受体拮抗剂对黑质多巴胺神经元兴奋性毒性损伤的调节作用。主要结果:1.Fluoro-Jade C(FJC)是新出现的荧光染料,与Fluoro-Jade与Fluoro-Jade B的性质相似。我们将FJC染色应用于MPTP和KA损伤小鼠模型,证明FJC染色可以成功显示黑质神经元的变性死亡,是一种简单、敏感、可靠的方法。FJC染色可清晰地显示黑质变性神经元的细胞胞体和突起。在中脑组织切片上,MPTP模型与KA模型黑质致密部内有许多FJC染色阳性的变性神经元,而对照组动物黑质内未见FJC染色阳性的变性神经元分布。2.采用FJC染色显示变性神经元、TH免疫染色显示存活的多巴胺神经元等方法,分析比较不同处理组的黑质神经元兴奋性毒性损伤情况。结果表明:与单纯KA组相比,NK3受体拮抗剂SB218795与KA共同处理组动物黑质内FJC阳性变性神经元数目明显减少,其动物旋转症状减轻,而TH阳性多巴胺神经元数目明显增加。而单纯NK3受体拮抗剂SB218795处理,未见明显毒性效应。主要结论:1.FJC方法能够很好地显示MPTP和KA动物模型黑质内神经元的变性死亡,具备很高的对比度和分辨率,是一种特异性标记黑质变性神经元树突、轴突和胞体的优良染色方法。2.神经激肽NK3受体拮抗剂能够有效地减轻或抑制KA诱导的黑质多巴胺神经元兴奋性毒性损伤与死亡速度,具有一定的神经保护作用。并进一步证明了神经激肽NK3的兴奋性毒性协同效应。本研究结果提示神经激肽-受体信号可能是参与PD病理发生的重要因素。神经激肽NK3受体作为一种新的药物干预靶位,具有应用于黑质神经元保护、完善PD治疗的潜在价值。更多还原

【Abstract】 Parkinson’s disease (PD) is one of most common neurodegenerative diseases, which is characterized by serious motor sympotoms such as resting tremor, bradykinesia, rigidity, gait disturbance and postural instability. PD is typically resulted from the massive degenerative death of dopamine neurons in the substantia nigra. Current treatments can not effectively prevent nigral neurons from progressive death and pathogical progress of PD. Previous studies have shown that glutamate-driving excitotoxicity may be one of crucial factors implicating in degeneration of nigral neurons and pathogenesis of PD. It is necessary, thus, to investigate novel strategies on preventing nigral dapamine neurons from excitotoxic lesions and improving the treatment of PD.Mammalian neurokinins are a family of tachykinin peptides that include substance P (neurokinin-1, NK-1), neurokinin A (NK-2) and neurokinin B (NK-3). Their biological functions are mediated by three distinct G-protein coupled neurokinin receptors, namely SP receptor (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 basal ganglia regions and known to significantly interact with neurons of the basal ganglia. Previous evidences had suggested that they may be involved in the regulation of physiological and pathological processes in the basal ganglia circuitry. In previous studies we had found that neurokinin agonists had intervention effects on exitotoxic injuries of nigral dopamine neurons of mice, particularly, administration of NK3 receptor agonist had neurotoxic synergistic effect. Further study is devoted to confirm the synergistic effect of NK3 on excitotoxicity and degenerative death of nigral dopamine neurons.In the present study, MPTP or KA-induced animal models, Fluoro-Jade C stain, administration of neuronkinin NK3 receptor antagonist, double immunofluorescence, and behavior analysis were applied to confirm effectiveness of FJC staining in the MPTP or KA-lesioned animal models and intervention role of NK3 receptor antagonist in exitotoxic injuries of nigral neurons.Firstly, Fluoro-Jade C stain can be effectively applied to detect the neuronal degeneration in the substantia nigra of animals induced by MPTP or KA insults, which shows higher resolution and contrast to stain the fine dendrites, axons and cell bodies of the degenerative neurons in the midbrain substantia nigra. In the midbrain tissue sections, FJC-positive degenerative neurons were numerously detected in the substantia nigra pars compacta, whereas they were not seen in the nigral region of control animals.Secondly, NK3 receptor antagonist effectively decreased KA-induced exitotoxic injuries of nigral dopamine neurons. It revealed that comparison with that of single KA-treated group, pretreatment with NK3 receptor antagonist relieved symptom of animal turning behavior, decreased numbers of FJC-positive degenerative neurons and increased numbers of TH-positive neurons. The data have implied that NK3 receptor antagonist may have certain neuroprotection on dopamine neurons.Taken together with previous observations, the present results have indicated that neurokinin NK3 signals may play an important role in excitotoxic degeneration of nigral dopamine neurons and pathogenesis of PD. This study also further suggests that NK3 receptor may function as potential therapeutic intervention targets in protecting nigral dopamine neurons and improving treatment of PD in human beings.更多还原