哺乳期CD-1 母鼠锌缺乏导致仔鼠海马神经元凋亡及其机制
发布时间：2018-07-24 14:38
【摘要】： 前言 锌是机体内的重要微量元素,在基因表达、DNA合成、酶催化反应、组织修复、神经信号传导以及记忆形成中发挥作用。脑中所含有的大部分锌离子(大约85%)与蛋白质等大分子相结合,如锌指蛋白和金属硫蛋白等;其余(大约15%)的锌离子则分布于含锌神经元轴突终末的突触前小泡中,这种游离锌离子作为神经调质被释放到突触间隙作用于突触后的受体,包括NMDA、AMPA/KA和GABA受体,调节神经信号传导。现已证明锌离子在突触可塑性中发挥特定的作用并参与学习与记忆功能的调节。 锌缺乏能够引起中枢神经系统内神经细胞凋亡,导致大脑学习记忆功能的损伤,但是其机制还不明了。研究表明神经元的存活和死亡与BDNF及其受体TrkB的表达和活性密切相关,而含锌神经元轴突终末的“突触小泡锌”可以通过两条途径调节TrkB的活性,即BDNF/TrkB和Src/TrkB途径,而TrkB的下游信号ERK可以调控多种凋亡相关蛋白,包括Bcl-2,Bax,Caspase-3等,从而参与神经细胞凋亡。因此,本研究以BDNF及其受体TrkB通路为切入点,探讨锌缺乏导致锌缺乏仔鼠海马神经元凋亡的可能机制。 材料与方法 待产CD-1母鼠随机分到锌缺乏组和对照组(n=6),分笼饲养。仔鼠出生后,母鼠和仔鼠同笼饲养,锌缺乏组母鼠产后即喂以缺锌饲料(锌含量为0.85ppm)以及去离子水;对照组母鼠则给予正常饮食(锌含量为30ppm)和去离子水。在小鼠生后第7d、14d、21d分别取两组仔鼠大脑,应用金属自显影技术(autometallography,AMG)、Nissl染色、TUNEL染色和Western blot技术检测锌缺乏仔鼠海马中游离锌离子含量变化、神经元的凋亡情况以及BDNF/TrkB通路相关蛋白水平的变化。 实验结果 AMG结果显示,在仔鼠未断乳期间锌缺乏组小鼠海马游离锌离子含量明显少于同龄正常对照组。Nissl染色以及TUNEL染色结果显示,哺乳期母鼠的锌缺乏导致了仔鼠海马神经元缺失和凋亡增加。Western Blot结果显示在小鼠生后第7d,14d,21d时:pro-BDNF(28kDa)和BDNF(14kDa)在锌缺乏组仔鼠海马中表达明显高于对照组;锌缺乏组仔鼠海马Src蛋白水平与对照组相比无显著差异,但Src自身抑制位点(Y527)磷酸化水平在锌缺乏组小鼠海马的表达明显增高;锌缺乏组TrkB(92kDa)的总量没有发生变化,但其磷酸化的p-TrkB(140kDa)明显减于对照组;细胞外受体蛋白激酶(extracellular receptor kinases,ERK)包括两种异构体ERK1和ERK2(分别为P44和P42),是将信号从表面受体传导至细胞核的关键。锌缺乏组仔鼠海马ERK总量无变化,但ERK磷酸化水平在锌缺乏组小鼠海马的表达明显降低。凋亡相关蛋白检测发现Bax/Bcl-2、Caspase-3在锌缺乏组仔鼠海马的表达明显增高。 结论 哺乳期母鼠锌缺乏会导致仔鼠海马中游离锌离子含量减少以及神经元凋亡的增加。锌缺乏抑制Src/TrkB/ERK途径导致促凋亡蛋白(包括Bax和Caspase-3)表达增强以及抗凋亡蛋白(Bcl-2)减少,而锌缺乏时的BDNF表达上调可能是一种不能完全代偿的保护性反应。
[Abstract]:Preface
Zinc is an important trace element in the body. It plays a role in gene expression, DNA synthesis, enzyme catalysis, tissue repair, neural signal transduction and memory formation. Most of the zinc ions in the brain (about 85%) are combined with proteins such as proteins such as zinc and metallothionein, and the rest (about 15%) of zinc ions are divided. In a presynaptic vesicle containing the end of the axon containing zinc neurons, this free zinc ion is released into the synaptic space to act on the postsynaptic receptors, including NMDA, AMPA/KA, and GABA receptors, to regulate nerve signal transduction. Adjust.
Zinc deficiency can cause neuronal apoptosis in the central nervous system and cause damage to the learning and memory function of the brain, but the mechanism is unknown. The study shows that the survival and death of the neurons are closely related to the expression and activity of BDNF and its receptor TrkB, and the "synaptic vesicle zinc" of the end of the axon containing zinc neurons can pass through two routes. The activity of TrkB, namely, the activity of BDNF/TrkB and Src/TrkB, and the downstream signal ERK of TrkB can regulate a variety of apoptosis related proteins, including Bcl-2, Bax, Caspase-3 and so on, so as to participate in the apoptosis of neurons. Therefore, this study uses BDNF and its receptor TrkB pathway as a breakthrough point to explore the possibility of zinc deficiency induced hippocampal neuronal apoptosis in zinc deficiency rats. Mechanism.
Materials and methods
CD-1 rats were randomly divided into zinc deficiency group and control group (n=6) and kept in cage. After birth, female rats and offspring were fed in the same cage. Zinc deficiency rats were fed with zinc deficiency diet (zinc content 0.85ppm) and deionized water after birth. The control rats were given normal diet (zinc content 30ppm) and deionized water. After birth, the mice were born 7d, 14d, 21d. Two groups of rat brains were taken respectively by metal autography (autometallography, AMG), Nissl staining, TUNEL staining and Western blot technique to detect the changes in the content of zinc ions in the middle reaches of the hippocampus, the apoptosis of neurons and the changes in the level of the protein related proteins of the BDNF/TrkB pathway in the hippocampus of the zinc deficiency rats.
experimental result
AMG results showed that the content of free zinc ions in the hippocampus of mice with zinc deficiency was significantly less than that of normal control group.Nissl staining and TUNEL staining results showed that zinc deficiency in lactating mice resulted in the loss of hippocampal neurons and apoptosis by.Western Blot in the breast-feeding mice, and the results showed that 7d, 14d, 21d in mice were at 7d, 14d and 21d: pro-. The expression of BDNF (28kDa) and BDNF (14kDa) in the hippocampus of zinc deficiency rats was significantly higher than that in the control group, but there was no significant difference in the level of Src protein in the hippocampus of the zinc deficiency rats, but the phosphorylation level of the Src self inhibition site (Y527) was significantly higher in the hippocampus of the zinc deficiency mice, and the total amount of TrkB (92kDa) in the zinc deficiency group did not change. The phosphorylation of p-TrkB (140kDa) was significantly reduced in the control group, and the extracellular receptor protein kinase (extracellular receptor kinases, ERK), including two isomers, ERK1 and ERK2 (P44 and P42), was the key to conducting the signal from the surface receptor to the nucleus. The total amount of the ERK in the hippocampus of the zinc deficient mice was not changed, but the phosphorylation level of the ERK was in zinc. The expression of Bax/Bcl-2 and Caspase-3 in the hippocampus of zinc deficiency mice was significantly decreased.
conclusion
Zinc deficiency in breast-feeding mice can lead to a decrease in the content of zinc ions in the middle reaches of the hippocampus and the increase of neuronal apoptosis in the hippocampus. Zinc deficiency inhibits the Src/TrkB/ERK pathway to increase the expression of apoptotic protein (including Bax and Caspase-3) and the decrease of apoptosis protein (Bcl-2), while the up regulation of BDNF expression in the zinc deficiency may be a kind of non complete compensation. A protective response.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363
[Abstract]:Preface
Zinc is an important trace element in the body. It plays a role in gene expression, DNA synthesis, enzyme catalysis, tissue repair, neural signal transduction and memory formation. Most of the zinc ions in the brain (about 85%) are combined with proteins such as proteins such as zinc and metallothionein, and the rest (about 15%) of zinc ions are divided. In a presynaptic vesicle containing the end of the axon containing zinc neurons, this free zinc ion is released into the synaptic space to act on the postsynaptic receptors, including NMDA, AMPA/KA, and GABA receptors, to regulate nerve signal transduction. Adjust.
Zinc deficiency can cause neuronal apoptosis in the central nervous system and cause damage to the learning and memory function of the brain, but the mechanism is unknown. The study shows that the survival and death of the neurons are closely related to the expression and activity of BDNF and its receptor TrkB, and the "synaptic vesicle zinc" of the end of the axon containing zinc neurons can pass through two routes. The activity of TrkB, namely, the activity of BDNF/TrkB and Src/TrkB, and the downstream signal ERK of TrkB can regulate a variety of apoptosis related proteins, including Bcl-2, Bax, Caspase-3 and so on, so as to participate in the apoptosis of neurons. Therefore, this study uses BDNF and its receptor TrkB pathway as a breakthrough point to explore the possibility of zinc deficiency induced hippocampal neuronal apoptosis in zinc deficiency rats. Mechanism.
Materials and methods
CD-1 rats were randomly divided into zinc deficiency group and control group (n=6) and kept in cage. After birth, female rats and offspring were fed in the same cage. Zinc deficiency rats were fed with zinc deficiency diet (zinc content 0.85ppm) and deionized water after birth. The control rats were given normal diet (zinc content 30ppm) and deionized water. After birth, the mice were born 7d, 14d, 21d. Two groups of rat brains were taken respectively by metal autography (autometallography, AMG), Nissl staining, TUNEL staining and Western blot technique to detect the changes in the content of zinc ions in the middle reaches of the hippocampus, the apoptosis of neurons and the changes in the level of the protein related proteins of the BDNF/TrkB pathway in the hippocampus of the zinc deficiency rats.
experimental result
AMG results showed that the content of free zinc ions in the hippocampus of mice with zinc deficiency was significantly less than that of normal control group.Nissl staining and TUNEL staining results showed that zinc deficiency in lactating mice resulted in the loss of hippocampal neurons and apoptosis by.Western Blot in the breast-feeding mice, and the results showed that 7d, 14d, 21d in mice were at 7d, 14d and 21d: pro-. The expression of BDNF (28kDa) and BDNF (14kDa) in the hippocampus of zinc deficiency rats was significantly higher than that in the control group, but there was no significant difference in the level of Src protein in the hippocampus of the zinc deficiency rats, but the phosphorylation level of the Src self inhibition site (Y527) was significantly higher in the hippocampus of the zinc deficiency mice, and the total amount of TrkB (92kDa) in the zinc deficiency group did not change. The phosphorylation of p-TrkB (140kDa) was significantly reduced in the control group, and the extracellular receptor protein kinase (extracellular receptor kinases, ERK), including two isomers, ERK1 and ERK2 (P44 and P42), was the key to conducting the signal from the surface receptor to the nucleus. The total amount of the ERK in the hippocampus of the zinc deficient mice was not changed, but the phosphorylation level of the ERK was in zinc. The expression of Bax/Bcl-2 and Caspase-3 in the hippocampus of zinc deficiency mice was significantly decreased.
conclusion
Zinc deficiency in breast-feeding mice can lead to a decrease in the content of zinc ions in the middle reaches of the hippocampus and the increase of neuronal apoptosis in the hippocampus. Zinc deficiency inhibits the Src/TrkB/ERK pathway to increase the expression of apoptotic protein (including Bax and Caspase-3) and the decrease of apoptosis protein (Bcl-2), while the up regulation of BDNF expression in the zinc deficiency may be a kind of non complete compensation. A protective response.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363
【共引文献】
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