缺氧时脑内神经元保护性相关蛋白的调节
发布时间：2018-09-10 06:59
【摘要】： 缺氧是临床常见病理现象,可导致多种缺氧性疾病,涉及呼吸、神经、心血管等诸多领域。由于脑组织储能少、耗氧量大,极易产生缺氧性损伤。因此,缺氧时的脑保护,是神经学研究的重要课题。 脑组织缺氧后,其细胞内ATP生成不足、酸中毒、细胞膜离子转运障碍、膜电位改变等,使神经递质合成减少及受体表达异常,这些功能蛋白的异常,影响了突触传递,导致脑细胞功能和结构改变。 氨基酸类神经递质是脑内重要的递质系统。脑细胞维持正常功能有赖于其中兴奋性氨基酸和抑制性氨基酸相互平衡。前者以谷氨酸和天冬氨酸为代表,介导大部分快速兴奋性突触传递,后者以γ-氨基丁酸(GABA)和甘氨酸(glycine)为代表,介导大部分快速抑制性突触传递。近来发现,抑制性氨基酸在保护脑细胞、抵抗缺氧/缺血和其它应激反应中发挥了重要作用,且抑制性氨基酸对不同发育期的神经元有不同的作用。然而,在缺氧环境中,不同年龄神经元的GABA受体和glycine受体的表达情况以及抑制性氨基酸对这两个受体表达的影响还不很清楚。 脑内另一重要的递质系统是乙酰胆碱系统,该系统参与了机体运动与感觉过程的信息传递。由于胆碱乙酰转移酶(ChAT)是乙酰胆碱合成的限速酶,它的异常与阿尔茨海默病(Alzheimer's disease,AD)等疾病密切相关。已经发现,缺氧影响了乙酰胆碱的代谢,参与了阿尔茨海默病的发生、发展,但缺氧究竟影响了乙酰胆碱系统的哪些环节,还有待进一步阐明。 本课题第一部分通过大鼠脑皮层神经元培养并予缺氧处理,在培养液中分别添加抑制性氨基酸γ-氨基丁酸、甘氨酸和牛磺酸进行刺激,测定GABA受体(GABA_ARα1)和glycine受体(GlyRα1)密度,以研究抑制性氨基酸对这两种受体的影响。 另外,文献报道,δ阿片受体(δ-opioid receptor,DOR)对脑缺氧/缺血具有保护作用。本课题第二、第三部分借助于本实验室建立的DOR转基因小鼠,经缺氧处理后,观察大脑皮层神经元中GABA受体及ChAT表达的变化,探讨缺氧和转基因DOR对GABA受体及ChAT的影响。 第一部分抑制性氨基酸对发育中缺氧性皮质神经元γ-氨基丁酸和甘氨酸受体表达影响的研究 目的:研究抑制性氨基酸对发育中缺氧性皮层神经元γ-氨基丁酸和甘氨酸受体表达的影响。 方法:取妊娠17-18天的胚胎大鼠脑皮层神经元原代培养,给予1%氧进行缺氧处理;在细胞培养液中,分别用不同浓度(10-2000μM)的抑制性氨基酸γ-氨基丁酸(GABA)、甘氨酸(glycine)和牛磺酸(taurine)于体外培养的第4天(DIV4)和20天(DIV20)两个时间点进行添加刺激;此外,分别在DIV4和DIV20时使用脑啡肽(DADLE,一种DOR激动剂)刺激。用Western blot方法测定细胞中GABA_ARα1和GlyRα1蛋白密度。 结果:(1)在体外生长发育的皮层神经元中,GABA_ARα1和GlyRα1的表达有变化,即在未发育成熟(DIV4)神经元中,GABA_ARα1表达较低,随着培养时间的延长,表达逐渐增加;GlyRα1表达在神经元发育早期已达成熟期的较高水平。(2)缺氧提高了未成熟神经元GABA_ARα1和GlyRα1的表达,而降低了GABA_ARα1和GlyRα1在成熟神经元的表达。(3)常氧状态下,GABA和glycine倾向于降低未成熟神经元GABA_ARα1和GlyRα1表达,但促进了发育成熟神经元的表达。(4)缺氧时,GABA,glycine和taurine降低了GABA_ARα1和GlyRα1在未成熟神经元中的表达,而对成熟神经元,则抵御了缺氧引起的受体表达下降。(5)δ阿片受体(δ-opioid receptor,DOR)的激活促进了GABA_ARα1和GlyRα1在常氧状态下成熟和未成熟神经元以及缺氧状态下成熟神经元中的表达。 结论:抑制性氨基酸对未成熟神经元由于其细胞毒性作用,降低了GABA_ARα1和GlyRα1在常氧和缺氧时的表达;而对成熟神经元,防止了缺氧引起的受体表达下降,并在常氧时保护了皮层细胞,同时上调了GABA_ARα1和GlyRα1表达;DOR有细胞保护作用,并提高了GABA_ARα1和GlyRα1表达。 第二部分慢性缺氧对DOR转基因小鼠脑内胆碱乙酰转移酶(ChAT)影响的研究 目的:用转基因增加DOR(δ-opioid receptor)表达的方式,研究慢性缺氧状态下小鼠脑内胆碱乙酰转移酶(ChAT)表达的变化,了解DOR对胆碱能神经元的作用。 方法:对成年(30日龄)和发育晚期(20日龄)之DOR转基因和野生型小鼠分别进行1天、3天、5天和7天缺氧(10%氧浓度)处理,取其皮层、皮层下、海马、脑干和小脑5个部位脑组织,用Western blot方法测定ChAT水平。 结果:在常氧环境中,DOR转基因鼠4个脑区(皮层、皮层下、海马、脑干)中ChAT密度明显低于野生型鼠;慢性缺氧(7天)使野生型鼠脑皮层、海马和脑干内ChAT密度降低,而同样慢性缺氧后,DOR转基因鼠上述脑区ChAT密度不但不下降,相反有增加趋势。 结论:缺氧降低了野生型小鼠脑内ChAT表达水平。而在转基因小鼠,由于DOR的高表达,防止了缺氧诱发的ChAT下降,提示DOR对缺氧状态下的胆碱能神经元具有保护作用。 第三部分缺氧对DOR转基因小鼠脑皮层神经元GABA_A受体表达影响的研究 目的:通过对DOR(δ-opioid receptor)转基因小鼠和野生型小鼠在缺氧和常氧状态下皮层神经元中GABA_A受体的研究,从整体动物水平了解缺氧和转基因DOR对大脑皮层神经元GABAA受体表达的影响,进一步探索DOR的神经保护作用。 方法:将DOR转基因小鼠和野生型小鼠分为缺氧组和常氧组(非缺氧组),对缺氧组分别施以1天、3天、5天和7天缺氧(10%氧浓度)处理。取其大脑皮层组织,提取蛋白质,周Western blot方法测定其GABA_A受体(GABA_ARα1)水平。 结果:在常氧环境,DOR转基因小鼠大脑皮层中GABA_ARα1密度倾向于较野生型鼠为低,慢性缺氧后这种差异更加明显(P＜0.05);经过1—7天的缺氧,野生型小鼠大脑皮层中GABA_ARα1表达无明显变化。 结论:转基因小鼠脑皮层中GABA_ARα1表达低于野生型小鼠;慢性缺氧进一步下调了转基因小鼠脑皮层神经元之GABA_ARα1表达;转基因DOR的存在,可能抑制了小鼠脑皮层神经元中GABA_ARα1的表达;7天时间的缺氧未能引起野生型小鼠脑内GABA_ARα1表达变化。
[Abstract]:Hypoxia is a common clinical pathological phenomenon, which can lead to a variety of hypoxic diseases, involving respiratory, nervous, cardiovascular and many other fields.
After brain tissue hypoxia, ATP production is insufficient, acidosis, membrane ion transport disorders, membrane potential changes and so on, resulting in decreased neurotransmitter synthesis and abnormal receptor expression. These functional proteins affect synaptic transmission, leading to changes in brain cell function and structure.
Amino acid neurotransmitters are important neurotransmitters in the brain.The normal function of brain cells depends on the balance between excitatory and inhibitory amino acids.Glutamic acid and aspartate are the representatives of the former,which mediate most of the rapid excitatory synaptic transmission,while GABA and glycine are the representatives of the latter. Recent studies have shown that inhibitory amino acids play an important role in protecting brain cells against hypoxia/ischemia and other stress responses, and that inhibitory amino acids play different roles in neurons of different developmental stages. The expression of the body and the effect of inhibitory amino acids on the expression of these two receptors are not clear.
Another important transmitter system in the brain is the acetylcholine system, which is involved in the transmission of information between movement and sensory processes. Because choline acetyltransferase (ChAT) is the rate-limiting enzyme for acetylcholine synthesis, its abnormalities are closely related to disease s such as Alzheimer's disease (AD). Alkali metabolism is involved in the occurrence and development of Alzheimer's disease, but what links of the acetylcholine system are affected by hypoxia remains to be further clarified.
In the first part of this study, the inhibiting amino acids gamma-aminobutyric acid, glycine and taurine were added to the culture medium to stimulate the cultured cortical neurons of rats. The density of GABA receptor (GABA_AR_ alpha1) and glycine receptor (GlyR_ alpha1) were measured to study the effect of inhibiting amino acids on the two receptors.
In addition, it has been reported that delta-opioid receptor (DOR) has protective effects on cerebral hypoxia/ischemia. In the second and third part of this study, the expression of GABA receptor and ChAT in cerebral cortex neurons was observed in hypoxic-treated DOR transgenic mice, and the effects of hypoxia and transgenic DOR on GABA receptor and C-hAT were investigated. The impact of hAT.
Part I Effects of Inhibitory Amino Acids on the Expression of Gamma-aminobutyric Acid and Glycine Receptors in Developing Hypoxic Cortical Neurons
AIM: To investigate the effects of inhibitory amino acids on the expression of gamma-aminobutyric acid and glycine receptors in developing hypoxic cortical neurons.
METHODS: The primary cultured cortical neurons of embryonic rats from 17 to 18 days of gestation were given 1% oxygen for hypoxia treatment, and the inhibitory amino acids gamma-aminobutyric acid (GABA), glycine and taurine (GABA) of different concentrations (10-2000 mu M) were used in the culture medium for the 4th day (DIV4) and 20th day (DIV20) respectively. In addition, enkephalin (DADLE, a DOR agonist) was used to stimulate DIV4 and DIV20, respectively. The density of GABA_AR_ alpha1 and GlyR_ alpha1 proteins was determined by Western blot.
Results: (1) The expression of GABA_AR_ alpha1 and GlyR_ alpha1 was changed in cortical neurons which grew in vitro, that is, the expression of GABA_AR_ alpha1 was lower in immature (DIV4) neurons, and increased gradually with the prolongation of culture time; the expression of GlyR_ alpha1 reached a higher level in the early stage of neuronal development. (2) Hypoxia increased the immature (DIV4) The expression of GABA_AR_ alpha1 increased gradually. The expression of GABA_AR alpha1 and GlyR alpha1 in neurons decreased, while the expression of GABA_AR alpha1 and GlyR alpha1 in mature neurons decreased. (3) Under normoxia, GABA and glycine tended to decrease the expression of GABA_AR alpha1 and GlyR alpha1 in immature neurons, but promoted the expression of mature neurons. (4) Under hypoxia, GABA, glycine and taurine decreased the expression of GABA_AR alpha1 and GlyR alpha1 in mature neurons. (5) The activation of delta-opioid receptor (DOR) promotes the expression of GABA_AR_ alpha 1 and GlyR_ alpha 1 in mature and immature neurons under normoxia and hypoxia.
CONCLUSION: Inhibitory amino acids can decrease the expression of GABA_AR_ alpha1 and GlyR_ alpha1 in immature neurons under normoxia and hypoxia due to their cytotoxic effects, while DOR can protect mature neurons from hypoxia-induced decrease of receptor expression and protect cortical cells under normoxia, and up-regulate the expression of GABA_AR_ alpha1 and GlyR_ alpha1. The expression of GABA_AR alpha 1 and GlyR alpha 1 was increased.
The second part is the effect of chronic hypoxia on the brain cholinyl acetyltransferase (ChAT) in DOR transgenic mice.
AIM: To study the expression of cholinergic acetyltransferase (ChAT) in the brain of mice under chronic hypoxia and to understand the effect of DOR on cholinergic neurons.
METHODS: Hypoxia (10% oxygen concentration) was performed on day 1, day 3, day 5 and day 7 in adult (30 days old) and late developmental (20 days old) DOR transgenic and wild type mice, respectively. The levels of ChAT in cortex, subcortex, hippocampus, brainstem and cerebellum were measured by Western blot.
Results: The ChAT density in four brain regions (cortex, subcortex, hippocampus and brainstem) of DOR transgenic mice was significantly lower than that of wild type mice in normoxic environment; the ChAT density in the cortex, hippocampus and brainstem of wild type mice was decreased by chronic hypoxia (7 days), but the ChAT density in the above brain regions of DOR transgenic mice did not decrease after the same chronic hypoxia.
CONCLUSION: Hypoxia decreases the expression of ChAT in the brain of wild type mice, but in transgenic mice, the high expression of DOR prevents hypoxia-induced decrease of ChAT, suggesting that DOR has protective effect on cholinergic neurons under hypoxia.
The third part is the effect of hypoxia on the expression of GABA_A receptor in cortical neurons of DOR transgenic mice.
AIM: To investigate the effects of hypoxia and transgenic DOR on the expression of GABAA receptors in cerebral cortical neurons and explore the neuroprotective effect of DOR.
Methods: DOR transgenic mice and wild type mice were divided into hypoxic group and normoxic group (non-hypoxic group). The hypoxic group was treated with hypoxia (10% oxygen concentration) for 1 day, 3 days, 5 days and 7 days, respectively. The cerebral cortex was extracted and the levels of GABA_A receptor (GABA_AR alpha 1) were determined by Western blot.
Results: In normal oxygen environment, the density of GABA_AR alpha 1 in the cerebral cortex of DOR transgenic mice tended to be lower than that of wild type mice, and the difference was more obvious after chronic hypoxia (P < 0.05).
CONCLUSION: The expression of GABA_AR alpha1 in the cerebral cortex of transgenic mice is lower than that of wild-type mice; chronic hypoxia further down-regulates the expression of GABA_AR alpha1 in the cerebral cortex of transgenic mice; the presence of transgenic DOR may inhibit the expression of GABA_AR alpha1 in the cerebral cortex neurons of mice; 7-day hypoxia does not induce GABA_AR alpha1 in the brain of wild-type mice. The expression of alpha 1 was changed.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363
本文编号：2233720
[Abstract]:Hypoxia is a common clinical pathological phenomenon, which can lead to a variety of hypoxic diseases, involving respiratory, nervous, cardiovascular and many other fields.
After brain tissue hypoxia, ATP production is insufficient, acidosis, membrane ion transport disorders, membrane potential changes and so on, resulting in decreased neurotransmitter synthesis and abnormal receptor expression. These functional proteins affect synaptic transmission, leading to changes in brain cell function and structure.
Amino acid neurotransmitters are important neurotransmitters in the brain.The normal function of brain cells depends on the balance between excitatory and inhibitory amino acids.Glutamic acid and aspartate are the representatives of the former,which mediate most of the rapid excitatory synaptic transmission,while GABA and glycine are the representatives of the latter. Recent studies have shown that inhibitory amino acids play an important role in protecting brain cells against hypoxia/ischemia and other stress responses, and that inhibitory amino acids play different roles in neurons of different developmental stages. The expression of the body and the effect of inhibitory amino acids on the expression of these two receptors are not clear.
Another important transmitter system in the brain is the acetylcholine system, which is involved in the transmission of information between movement and sensory processes. Because choline acetyltransferase (ChAT) is the rate-limiting enzyme for acetylcholine synthesis, its abnormalities are closely related to disease s such as Alzheimer's disease (AD). Alkali metabolism is involved in the occurrence and development of Alzheimer's disease, but what links of the acetylcholine system are affected by hypoxia remains to be further clarified.
In the first part of this study, the inhibiting amino acids gamma-aminobutyric acid, glycine and taurine were added to the culture medium to stimulate the cultured cortical neurons of rats. The density of GABA receptor (GABA_AR_ alpha1) and glycine receptor (GlyR_ alpha1) were measured to study the effect of inhibiting amino acids on the two receptors.
In addition, it has been reported that delta-opioid receptor (DOR) has protective effects on cerebral hypoxia/ischemia. In the second and third part of this study, the expression of GABA receptor and ChAT in cerebral cortex neurons was observed in hypoxic-treated DOR transgenic mice, and the effects of hypoxia and transgenic DOR on GABA receptor and C-hAT were investigated. The impact of hAT.
Part I Effects of Inhibitory Amino Acids on the Expression of Gamma-aminobutyric Acid and Glycine Receptors in Developing Hypoxic Cortical Neurons
AIM: To investigate the effects of inhibitory amino acids on the expression of gamma-aminobutyric acid and glycine receptors in developing hypoxic cortical neurons.
METHODS: The primary cultured cortical neurons of embryonic rats from 17 to 18 days of gestation were given 1% oxygen for hypoxia treatment, and the inhibitory amino acids gamma-aminobutyric acid (GABA), glycine and taurine (GABA) of different concentrations (10-2000 mu M) were used in the culture medium for the 4th day (DIV4) and 20th day (DIV20) respectively. In addition, enkephalin (DADLE, a DOR agonist) was used to stimulate DIV4 and DIV20, respectively. The density of GABA_AR_ alpha1 and GlyR_ alpha1 proteins was determined by Western blot.
Results: (1) The expression of GABA_AR_ alpha1 and GlyR_ alpha1 was changed in cortical neurons which grew in vitro, that is, the expression of GABA_AR_ alpha1 was lower in immature (DIV4) neurons, and increased gradually with the prolongation of culture time; the expression of GlyR_ alpha1 reached a higher level in the early stage of neuronal development. (2) Hypoxia increased the immature (DIV4) The expression of GABA_AR_ alpha1 increased gradually. The expression of GABA_AR alpha1 and GlyR alpha1 in neurons decreased, while the expression of GABA_AR alpha1 and GlyR alpha1 in mature neurons decreased. (3) Under normoxia, GABA and glycine tended to decrease the expression of GABA_AR alpha1 and GlyR alpha1 in immature neurons, but promoted the expression of mature neurons. (4) Under hypoxia, GABA, glycine and taurine decreased the expression of GABA_AR alpha1 and GlyR alpha1 in mature neurons. (5) The activation of delta-opioid receptor (DOR) promotes the expression of GABA_AR_ alpha 1 and GlyR_ alpha 1 in mature and immature neurons under normoxia and hypoxia.
CONCLUSION: Inhibitory amino acids can decrease the expression of GABA_AR_ alpha1 and GlyR_ alpha1 in immature neurons under normoxia and hypoxia due to their cytotoxic effects, while DOR can protect mature neurons from hypoxia-induced decrease of receptor expression and protect cortical cells under normoxia, and up-regulate the expression of GABA_AR_ alpha1 and GlyR_ alpha1. The expression of GABA_AR alpha 1 and GlyR alpha 1 was increased.
The second part is the effect of chronic hypoxia on the brain cholinyl acetyltransferase (ChAT) in DOR transgenic mice.
AIM: To study the expression of cholinergic acetyltransferase (ChAT) in the brain of mice under chronic hypoxia and to understand the effect of DOR on cholinergic neurons.
METHODS: Hypoxia (10% oxygen concentration) was performed on day 1, day 3, day 5 and day 7 in adult (30 days old) and late developmental (20 days old) DOR transgenic and wild type mice, respectively. The levels of ChAT in cortex, subcortex, hippocampus, brainstem and cerebellum were measured by Western blot.
Results: The ChAT density in four brain regions (cortex, subcortex, hippocampus and brainstem) of DOR transgenic mice was significantly lower than that of wild type mice in normoxic environment; the ChAT density in the cortex, hippocampus and brainstem of wild type mice was decreased by chronic hypoxia (7 days), but the ChAT density in the above brain regions of DOR transgenic mice did not decrease after the same chronic hypoxia.
CONCLUSION: Hypoxia decreases the expression of ChAT in the brain of wild type mice, but in transgenic mice, the high expression of DOR prevents hypoxia-induced decrease of ChAT, suggesting that DOR has protective effect on cholinergic neurons under hypoxia.
The third part is the effect of hypoxia on the expression of GABA_A receptor in cortical neurons of DOR transgenic mice.
AIM: To investigate the effects of hypoxia and transgenic DOR on the expression of GABAA receptors in cerebral cortical neurons and explore the neuroprotective effect of DOR.
Methods: DOR transgenic mice and wild type mice were divided into hypoxic group and normoxic group (non-hypoxic group). The hypoxic group was treated with hypoxia (10% oxygen concentration) for 1 day, 3 days, 5 days and 7 days, respectively. The cerebral cortex was extracted and the levels of GABA_A receptor (GABA_AR alpha 1) were determined by Western blot.
Results: In normal oxygen environment, the density of GABA_AR alpha 1 in the cerebral cortex of DOR transgenic mice tended to be lower than that of wild type mice, and the difference was more obvious after chronic hypoxia (P < 0.05).
CONCLUSION: The expression of GABA_AR alpha1 in the cerebral cortex of transgenic mice is lower than that of wild-type mice; chronic hypoxia further down-regulates the expression of GABA_AR alpha1 in the cerebral cortex of transgenic mice; the presence of transgenic DOR may inhibit the expression of GABA_AR alpha1 in the cerebral cortex neurons of mice; 7-day hypoxia does not induce GABA_AR alpha1 in the brain of wild-type mice. The expression of alpha 1 was changed.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363
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3 甘平,杨茹,程介士;GABA受体在脑缺血中的作用[J];中国神经科学杂志;2004年01期
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