碱性成纤维细胞生长因子对帕金森病神经递质的影响及其保护机制研究
发布时间：2018-11-28 09:22
【摘要】：帕金森病(Parkinson's disease, PD)是一种好发于中老年人的中枢神经退行性疾病,主要病理特征在于黑质-纹状体内多巴胺(DA)能神经元的变性,导致脑部神经递质DA缺乏。PD病变复杂,发病机制尚未完全阐明,普遍认为是一种与一些神经递质系统相关的可逐渐恶化的多中心神经变性疾病,其临床症状主要表现为静止性震颤、运动迟缓、肌僵直和姿势平衡障碍。帕金森病手术治疗昂贵,目前还是以药物治疗为主。但已有的抗帕金森病药物只起着改善症状的作用,不能延缓疾病的进程,不能预防DA能神经元的退变,且长期服用,其用量越来越大,副作用越来越多。近年来,帕金森病药物治疗由直接补充DA转向多环节治疗,重点集中于对DA能神经元的保护作用。目前许多研究者关注应用神经营养因子和生长因子治疗帕金森病,其中碱性成纤维细胞生长因子(bFGF)作为一类生长因子,具有促有丝分裂的作用,在人体组织上含有丰富的受体,在体内和体外均具有活性,主要通过与细胞表面的酪氨酸激酶型受体作用而发挥着广泛的生物学功能,在组织创伤修复和神经保护等方面起着重要的作用。 本论文从PD病变源头—神经元的营养修护角度,从动物行为学、神经递质含量、神经元数目等方面的变化来研究bFGF对PD脑内神经递质的影响,并通过检测多种相关生化指标初步来阐明bFGF修护神经元和影响PD神经递质可能的作用机制。具体内容包括以下三点： (1)建立了由神经毒素6-羟基多巴(6-OHDA)诱发的帕金森病细胞模型。在细胞水平上,bFGF能够提高帕金森病PC12模型细胞的成活率,降低6-OHDA引起的PC12细胞凋亡,促进细胞中相关单胺类神经递质及其代谢产物的分泌,刺激帕金森病模型细胞中酪氨酸羟化酶(TH)的表达。同时bFGF能提高帕金森病模型细胞中磷酸化Akt和ERK的蛋白水平,抑制细胞中内质网应激蛋白(CHOP、GRP78和CASPASE12)的表达,提示bFGF可能通过激活PI3K/Akt和ERK1/2通路来抑制6-OHDA氧化损伤引起的内质网应激,降低神经细胞凋亡,从而实现神经保护作用。 (2)采用脑立体定位注射6-OHDA构建了PD大鼠偏侧模型,并通过腹腔注射阿扑吗啡溶液引起的动物行为学来验证模型的成功。在动物水平上,bFGF可以有效改善模型动物的帕金森病症状；提高大鼠脑纹状体组织中细胞外液和细胞内外总的单胺类神经递质含量；促进脑部黑质和纹状体中TH的表达。此外,bFGF能促进PD大鼠脑内自由基的清除,上调PD大鼠损毁侧纹状体中磷酸化Akt和ERK的水平,抑制内质网应激蛋白(CHOP、GRP78和CASPASE12)以及α-Syn蛋白的表达,提示bFGF可能通过激活PI3K/Akt和ERK1/2通路,抑制6-OHDA氧化损伤引起的内质网应激和α-Syn蛋白水平,促使TH的表达提高,降低DA能神经元凋亡,增加神经递质的合成,最终表现出PD症状改善,实现帕金森病的神经保护作用。 (3)以基于1HNMR的代谢组学方法对PD大鼠脑纹状体进行研究,发现PD大鼠脑纹状体中乳酸、γ-氨基丁酸、谷氨酸、肌酸、甘氨酸和肌醇含量增加；N-乙酰天门冬氨酸、牛磺酸的含量减少。提示PD脑中形成了兴奋性毒性,参与了DA能神经元凋亡。经过bFGF治疗后,PD大鼠脑纹状体中γ-氨基丁酸、N-乙酰天门冬氨酸和牛磺酸的含量明显上升；谷氨酸、肌醇和乳酸的含量明显下降。表明bFGF能使纹状体中失衡的氨基酸类神经递质向常态恢复,改善星形胶质细胞增生,促进星形胶质细胞营养因子的分泌,对DA能神经元具有营养和修护作用。 总而言之,本论文研究了bFGF用药前后帕金森病神经递质的变化,以及内质网应激和a-Syn蛋白表达等生化指标变化来探讨bFGF对帕金森病的保护作用,为bFGF能改善PD症状提供了一个新的可阐明的机制,其研究结果可为设计新型的PD治疗药物和建立PD治疗策略提供实验依据。此外,首次采用代谢组学方法,从系统生物学层面上发现了PD脑中一些代谢异常的氨基酸类小分子,为帕金森病的发病机制、发展演化以及治疗研究提供新的研究思路。
[Abstract]:Parkinson's disease (PD) is a kind of central nervous system of the middle-aged and the elderly. The main pathological features are the degeneration of the dopaminergic neurons in the substantia nigra-striatal, which leads to the lack of the DA in the brain. PD is a complex disease, and the pathogenesis has not yet been fully set forth. It is generally believed to be a progressive, multi-center neurodegenerative disease associated with some neurotransmitter systems. The clinical symptoms of PD are mainly static tremor, bradykinesia, muscle stiffness and postural balance disorder. The treatment of Parkinson's disease is expensive, and is currently based on drug therapy. However, the anti-Parkinson's disease has the effect of improving the symptoms only, can not delay the process of the disease, can not prevent the detransformation of the DA energy neuron, and is taken for a long time, the dosage of the anti-Parkinson's disease drug is more and more large, and the side effect is more and more. In recent years, the treatment of Parkinson's disease is controlled by the direct supplement of DA to multi-link therapy, focusing on the protection of DA energy neurons. At present, many researchers are concerned about the application of neurotrophin and growth factors to the treatment of parkinson's disease, in which basic fibroblast growth factor (bFGF), as a kind of growth factor, has a mitogenic effect, In vivo and in vitro, the activity mainly plays an important role in tissue wound repair and neuroprotection by playing a wide range of biological functions through the role of the tyrosine kinase-like receptors on the surface of the cells. In this paper, the effects of bFGF on neurotransmitters in PD were studied from the aspects of animal behavior, neurotransmitter content and number of neurons from the perspective of the nutritional repair of the source of PD. In response, and through the detection of a variety of related biochemical indexes, the effect of the bFGF on the repair of the neurons and the potential of the PD neurotransmitters is explained. The specific contents of the system are as follows: Point: (1) The establishment of a fine Parkinson's disease induced by neurotoxin 6-hydroxydopamine (6-OHDA) In cell level, bFGF can improve the survival rate of PC12 model cells, decrease the apoptosis of PC12 cells induced by 6-OHDA, promote the secretion of the related monoamines and their metabolites in the cells, and stimulate the tyrosine hydroxylase (TH) in the model cells of Parkinson's disease. The expression of bFGF can improve the protein level of phosphorylated Akt and ERK in the model cells of Parkinson's disease, and inhibit the expression of endoplasmic reticulum stress protein (CHOP, GRP78 and CASPASE12) in the cells. It is suggested that bFGF may inhibit the endoplasmic reticulum stress caused by the oxidative damage of 6-OHDA by activating the PI3K/ Akt and ERK1/ 2 pathways, and reduce the nerve fine. Apoptosis, thus realizing neuroprotection (2) The partial side model of PD rats was constructed by stereotaxic injection of 6-OHDA and the behavior of animal behavior caused by injection of apomorphine in the abdominal cavity was verified. The success of the model is that, at the animal level, bFGF can effectively improve the symptoms of the Parkinson's disease in the model animals, improve the content of the monoamines in the extracellular fluid and the inside and outside the cells of the rat brain striatal tissue, and promote the substantia nigra and the striatum of the brain. In addition, bFGF can promote the clearance of free radicals in the brain of PD rats, increase the level of phosphorylated Akt and ERK in the rat striatum of PD rats, and inhibit the expression of endoplasmic reticulum stress protein (CHOP, GRP78 and CASPASE12), and the expression of HCO3-Syn protein, suggesting that bFGF may activate PI3K/ Akt and ERK by activating PI3K/ Akt and ERK. 1/ 2 pathway, to inhibit the endoplasmic reticulum stress and the P-Syn protein level caused by the 6-OHDA oxidative damage, to promote the expression of TH, to reduce the apoptosis of the DA and to increase the synthesis of the neurotransmitters, and finally to show the improvement of the PD symptoms and the realization of the God of the Parkinson's disease. (3) The brain striatum of PD rats was studied by means of a metabolic group based on 1HNMR, and the content of lactic acid, L-aminobutyric acid, glutamic acid, myoacid, glycine and inositol increased in the striatum of PD rats. The content of the sulfonic acid is reduced. It is suggested that the PD brain forms an excitable toxicity and is involved in D A. The content of P-aminobutyric acid, N-glycinate and taurine in the striatum of PD rats increased significantly after the treatment of bFGF. Glutamic acid, inositol and lactic acid The results showed that bFGF can restore the unbalanced amino acid neurotransmitters in the striatum to normal state, improve the proliferation of astrocytes, promote the secretion of the nutrient factors of astrocytes, and have the effects on the dopaminergic neurons. The effects of bFGF on the protective effect of bFGF on parkinson's disease were discussed in this paper. A new eluctable mechanism, the results of which can be designed to design new PD-treated drugs and to establish PD The experimental basis for the therapeutic strategy is provided. In addition, the metabolic group learning method is adopted for the first time, and the amino acid type small molecules of some metabolic abnormalities in the PD brain are found at the systemic biological level, and the pathogenesis, the development and the evolution of the Parkinson's disease and the treatment of the disease are developed.
【学位授予单位】：南京理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.5
本文编号：2362449
[Abstract]:Parkinson's disease (PD) is a kind of central nervous system of the middle-aged and the elderly. The main pathological features are the degeneration of the dopaminergic neurons in the substantia nigra-striatal, which leads to the lack of the DA in the brain. PD is a complex disease, and the pathogenesis has not yet been fully set forth. It is generally believed to be a progressive, multi-center neurodegenerative disease associated with some neurotransmitter systems. The clinical symptoms of PD are mainly static tremor, bradykinesia, muscle stiffness and postural balance disorder. The treatment of Parkinson's disease is expensive, and is currently based on drug therapy. However, the anti-Parkinson's disease has the effect of improving the symptoms only, can not delay the process of the disease, can not prevent the detransformation of the DA energy neuron, and is taken for a long time, the dosage of the anti-Parkinson's disease drug is more and more large, and the side effect is more and more. In recent years, the treatment of Parkinson's disease is controlled by the direct supplement of DA to multi-link therapy, focusing on the protection of DA energy neurons. At present, many researchers are concerned about the application of neurotrophin and growth factors to the treatment of parkinson's disease, in which basic fibroblast growth factor (bFGF), as a kind of growth factor, has a mitogenic effect, In vivo and in vitro, the activity mainly plays an important role in tissue wound repair and neuroprotection by playing a wide range of biological functions through the role of the tyrosine kinase-like receptors on the surface of the cells. In this paper, the effects of bFGF on neurotransmitters in PD were studied from the aspects of animal behavior, neurotransmitter content and number of neurons from the perspective of the nutritional repair of the source of PD. In response, and through the detection of a variety of related biochemical indexes, the effect of the bFGF on the repair of the neurons and the potential of the PD neurotransmitters is explained. The specific contents of the system are as follows: Point: (1) The establishment of a fine Parkinson's disease induced by neurotoxin 6-hydroxydopamine (6-OHDA) In cell level, bFGF can improve the survival rate of PC12 model cells, decrease the apoptosis of PC12 cells induced by 6-OHDA, promote the secretion of the related monoamines and their metabolites in the cells, and stimulate the tyrosine hydroxylase (TH) in the model cells of Parkinson's disease. The expression of bFGF can improve the protein level of phosphorylated Akt and ERK in the model cells of Parkinson's disease, and inhibit the expression of endoplasmic reticulum stress protein (CHOP, GRP78 and CASPASE12) in the cells. It is suggested that bFGF may inhibit the endoplasmic reticulum stress caused by the oxidative damage of 6-OHDA by activating the PI3K/ Akt and ERK1/ 2 pathways, and reduce the nerve fine. Apoptosis, thus realizing neuroprotection (2) The partial side model of PD rats was constructed by stereotaxic injection of 6-OHDA and the behavior of animal behavior caused by injection of apomorphine in the abdominal cavity was verified. The success of the model is that, at the animal level, bFGF can effectively improve the symptoms of the Parkinson's disease in the model animals, improve the content of the monoamines in the extracellular fluid and the inside and outside the cells of the rat brain striatal tissue, and promote the substantia nigra and the striatum of the brain. In addition, bFGF can promote the clearance of free radicals in the brain of PD rats, increase the level of phosphorylated Akt and ERK in the rat striatum of PD rats, and inhibit the expression of endoplasmic reticulum stress protein (CHOP, GRP78 and CASPASE12), and the expression of HCO3-Syn protein, suggesting that bFGF may activate PI3K/ Akt and ERK by activating PI3K/ Akt and ERK. 1/ 2 pathway, to inhibit the endoplasmic reticulum stress and the P-Syn protein level caused by the 6-OHDA oxidative damage, to promote the expression of TH, to reduce the apoptosis of the DA and to increase the synthesis of the neurotransmitters, and finally to show the improvement of the PD symptoms and the realization of the God of the Parkinson's disease. (3) The brain striatum of PD rats was studied by means of a metabolic group based on 1HNMR, and the content of lactic acid, L-aminobutyric acid, glutamic acid, myoacid, glycine and inositol increased in the striatum of PD rats. The content of the sulfonic acid is reduced. It is suggested that the PD brain forms an excitable toxicity and is involved in D A. The content of P-aminobutyric acid, N-glycinate and taurine in the striatum of PD rats increased significantly after the treatment of bFGF. Glutamic acid, inositol and lactic acid The results showed that bFGF can restore the unbalanced amino acid neurotransmitters in the striatum to normal state, improve the proliferation of astrocytes, promote the secretion of the nutrient factors of astrocytes, and have the effects on the dopaminergic neurons. The effects of bFGF on the protective effect of bFGF on parkinson's disease were discussed in this paper. A new eluctable mechanism, the results of which can be designed to design new PD-treated drugs and to establish PD The experimental basis for the therapeutic strategy is provided. In addition, the metabolic group learning method is adopted for the first time, and the amino acid type small molecules of some metabolic abnormalities in the PD brain are found at the systemic biological level, and the pathogenesis, the development and the evolution of the Parkinson's disease and the treatment of the disease are developed.
【学位授予单位】：南京理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.5
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