發(fā)布時間：2018-07-24 14:38

【摘要】： 前言 鋅是機體內(nèi)的重要微量元素,在基因表達、DNA合成、酶催化反應、組織修復、神經(jīng)信號傳導以及記憶形成中發(fā)揮作用。腦中所含有的大部分鋅離子(大約85%)與蛋白質(zhì)等大分子相結(jié)合,如鋅指蛋白和金屬硫蛋白等;其余(大約15%)的鋅離子則分布于含鋅神經(jīng)元軸突終末的突觸前小泡中,這種游離鋅離子作為神經(jīng)調(diào)質(zhì)被釋放到突觸間隙作用于突觸后的受體,包括NMDA、AMPA/KA和GABA受體,調(diào)節(jié)神經(jīng)信號傳導。現(xiàn)已證明鋅離子在突觸可塑性中發(fā)揮特定的作用并參與學習與記憶功能的調(diào)節(jié)。 鋅缺乏能夠引起中樞神經(jīng)系統(tǒng)內(nèi)神經(jīng)細胞凋亡,導致大腦學習記憶功能的損傷,但是其機制還不明了。研究表明神經(jīng)元的存活和死亡與BDNF及其受體TrkB的表達和活性密切相關(guān),而含鋅神經(jīng)元軸突終末的“突觸小泡鋅”可以通過兩條途徑調(diào)節(jié)TrkB的活性,即BDNF/TrkB和Src/TrkB途徑,而TrkB的下游信號ERK可以調(diào)控多種凋亡相關(guān)蛋白,包括Bcl-2,Bax,Caspase-3等,從而參與神經(jīng)細胞凋亡。因此,本研究以BDNF及其受體TrkB通路為切入點,探討鋅缺乏導致鋅缺乏仔鼠海馬神經(jīng)元凋亡的可能機制。 材料與方法 待產(chǎn)CD-1母鼠隨機分到鋅缺乏組和對照組(n=6),分籠飼養(yǎng)。仔鼠出生后,母鼠和仔鼠同籠飼養(yǎng),鋅缺乏組母鼠產(chǎn)后即喂以缺鋅飼料(鋅含量為0.85ppm)以及去離子水;對照組母鼠則給予正常飲食(鋅含量為30ppm)和去離子水。在小鼠生后第7d、14d、21d分別取兩組仔鼠大腦,應用金屬自顯影技術(shù)(autometallography,AMG)、Nissl染色、TUNEL染色和Western blot技術(shù)檢測鋅缺乏仔鼠海馬中游離鋅離子含量變化、神經(jīng)元的凋亡情況以及BDNF/TrkB通路相關(guān)蛋白水平的變化。 實驗結(jié)果 AMG結(jié)果顯示,在仔鼠未斷乳期間鋅缺乏組小鼠海馬游離鋅離子含量明顯少于同齡正常對照組。Nissl染色以及TUNEL染色結(jié)果顯示,哺乳期母鼠的鋅缺乏導致了仔鼠海馬神經(jīng)元缺失和凋亡增加。Western Blot結(jié)果顯示在小鼠生后第7d,14d,21d時:pro-BDNF(28kDa)和BDNF(14kDa)在鋅缺乏組仔鼠海馬中表達明顯高于對照組;鋅缺乏組仔鼠海馬Src蛋白水平與對照組相比無顯著差異,但Src自身抑制位點(Y527)磷酸化水平在鋅缺乏組小鼠海馬的表達明顯增高;鋅缺乏組TrkB(92kDa)的總量沒有發(fā)生變化,但其磷酸化的p-TrkB(140kDa)明顯減于對照組;細胞外受體蛋白激酶(extracellular receptor kinases,ERK)包括兩種異構(gòu)體ERK1和ERK2(分別為P44和P42),是將信號從表面受體傳導至細胞核的關(guān)鍵。鋅缺乏組仔鼠海馬ERK總量無變化,但ERK磷酸化水平在鋅缺乏組小鼠海馬的表達明顯降低。凋亡相關(guān)蛋白檢測發(fā)現(xiàn)Bax/Bcl-2、Caspase-3在鋅缺乏組仔鼠海馬的表達明顯增高。 結(jié)論 哺乳期母鼠鋅缺乏會導致仔鼠海馬中游離鋅離子含量減少以及神經(jīng)元凋亡的增加。鋅缺乏抑制Src/TrkB/ERK途徑導致促凋亡蛋白(包括Bax和Caspase-3)表達增強以及抗凋亡蛋白(Bcl-2)減少,而鋅缺乏時的BDNF表達上調(diào)可能是一種不能完全代償?shù)谋Ｗo性反應。
[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.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

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本文編號：2141692