【摘要】：目的：阿爾茨海默病(Alzheimer disease,AD),是一種中樞神經(jīng)系統(tǒng)退行性疾病。主要表現(xiàn)為進(jìn)行性記憶障礙、認(rèn)知功能障礙、人格改變及語(yǔ)言障礙等神經(jīng)精神癥狀,嚴(yán)重影響社交、職業(yè)與生活質(zhì)量。AD的病因及發(fā)病機(jī)制尚未闡明,特征性病理改變?yōu)閜淀粉樣蛋白(β-amyloid,Aβ)沉積形成的細(xì)胞外老年斑和Tau蛋白過(guò)度磷酸化形成的細(xì)胞內(nèi)神經(jīng)原纖維纏結(jié)。Ap的代謝前體為淀粉樣前體蛋白(Amyloid Precursor Protein, APP)。因其剪切產(chǎn)物Ap是構(gòu)成老年斑的主要成分,長(zhǎng)期以來(lái)人們對(duì)APP的研究大部分集中在其代謝途徑異常對(duì)AD發(fā)病機(jī)制的影響方面。APP作為一種管家基因在人體多種組織中均有表達(dá),但對(duì)其功能卻知之甚少,尤其APP對(duì)神經(jīng)元興奮性以及與電壓門控性鈉通道的功能聯(lián)系至今未見(jiàn)報(bào)道。盡管有報(bào)道稱Ap可影響神經(jīng)興奮,但多數(shù)研究集中在Ap提高谷氨酸興奮性毒性上,鮮有探討Ap影響鈉通道及神經(jīng)元本身的興奮性。本實(shí)驗(yàn)室前期實(shí)驗(yàn)結(jié)果顯示APP與電壓門控鈉通道特異地匯聚在中樞神經(jīng)系統(tǒng)郎飛氏結(jié)上,影響神經(jīng)傳導(dǎo),因此本研究的主要目的是從功能水平上進(jìn)一步研究APP及其產(chǎn)物Ap與神經(jīng)元興奮性和電壓門控鈉通道之間的關(guān)系,為揭示APP的生理功能及AD的發(fā)病機(jī)制提供新的理論依據(jù)。方法：APP/PS1轉(zhuǎn)基因鼠體外原代神經(jīng)元培養(yǎng),電流鉗探討神經(jīng)元興奮性的變化：APP轉(zhuǎn)染不同亞型電壓門控鈉通道(Nav1.6, Nav1.2)HEK 293細(xì)胞系,全細(xì)胞膜片鉗探討APP和鈉通道的功能聯(lián)系；Aβ_(1-42)干預(yù)體外原代培養(yǎng)神經(jīng)元,全細(xì)胞膜片鉗探討神經(jīng)元興奮性和電壓門控鈉通道的變化。結(jié)果：1.APP可提高神經(jīng)元?jiǎng)幼麟娢环逯怠?.APP可降低神經(jīng)元?jiǎng)幼麟娢坏拈撾娢弧?.APP可增加神經(jīng)元?jiǎng)幼麟娢坏陌l(fā)放頻率。4.APP可增大HEK 293 Nav1.6細(xì)胞系電流幅值。5.APP可加快HEK 293 Nav1.6鈉通道的激活。6.APP可減慢HEK 293 Nav1.6鈉通道的失活。7.APP對(duì)HEK 293 Nav1.2細(xì)胞系鈉通道沒(méi)有調(diào)控作用。8.Aβ_(1-42)可降低神經(jīng)元?jiǎng)幼麟娢坏拈撾娢弧?.Aβ_(1-42)可增加神經(jīng)元?jiǎng)幼麟娢坏陌l(fā)放頻率。10.Aβ_(1-42)對(duì)鈉通道電流似乎無(wú)影響。結(jié)論：1.APP過(guò)表達(dá)可提高神經(jīng)元興奮性。2.APP可特異增強(qiáng)Nav1.6通道的電流,而對(duì)Nav1.2電流無(wú)調(diào)控作用。3.Aβ_(1-42)亦可增加神經(jīng)元興奮性,但對(duì)鈉通道電流似乎無(wú)影響。
[Abstract]:Objective: Alzheimer's disease (Alzheimer disease,AD) is a degenerative disease of central nervous system. The main manifestations are progressive memory impairment, cognitive impairment, personality changes and language disorders, which seriously affect social interaction, occupation and quality of life. The etiology and pathogenesis of AD have not been clarified, and the characteristic pathological changes are p-amyloid,. A 尾) deposited extracellular plaques and intracellular neurofibrillar tangles formed by hyperphosphorylation of Tau protein. The metabolic precursor of AP is starch precursor protein (Amyloid Precursor Protein, APP). Because its shear product Ap is the main component of elderly plaques, most of the research on APP has been focused on the effect of abnormal metabolic pathway on the pathogenesis of AD. App, as a housekeeper gene, is expressed in a variety of human tissues, but little is known about its function, especially the relationship between APP and voltage-gated sodium channel has not been reported. Although it has been reported that Ap can affect nerve excitation, most of the studies have focused on the enhancement of glutamate excitotoxicity by Ap. Few studies have discussed the effects of Ap on sodium channels and the excitability of neurons themselves. The results of previous experiments in our laboratory show that APP and voltage-gated sodium channel converge specifically on Langfei's node of central nervous system, which affects nerve conduction. Therefore, the main purpose of this study is to further study the relationship between APP and its product Ap and neuron excitability and voltage-gated sodium channel at functional level, so as to provide a new theoretical basis for revealing the physiological function of APP and the pathogenesis of AD. Methods: the primary neurons of APP/PS1 transgenic mice were cultured in vitro, and the changes of neuron excitability were investigated by current clamp: APP was transfected into different subtypes of voltage gated sodium channel (Nav1.6, Nav1.2) HEK 293cell line, the functional relationship between APP and sodium channel was discussed by whole cell patch clamp, A 尾 _ (1 鈮，

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