王葉冉,劉雨輝,梁春榮,曾凡,卜先樂,矯樹生,王慶華,姚秀卿,周華東,王延江
阿爾茨海默病(Alzheimer disease,AD)是一種以皮質(zhì)、海馬神經(jīng)元丟失,淀粉樣β蛋白(amyloid β-protein,Aβ)聚集形成老年斑以及Tau蛋白過度磷酸化形成神經(jīng)纖維纏結(jié)為主要病理改變的中樞神經(jīng)系統(tǒng)變性疾病,是最常見的癡呆類型,臨床主要表現(xiàn)為進(jìn)行性的認(rèn)知功能減退,尤其是記憶力下降。p75神經(jīng)營(yíng)養(yǎng)因子受體(p75neurotrophin receptor,p75NTR)是腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)、神經(jīng)生長(zhǎng)因子(NGF)等神經(jīng)營(yíng)養(yǎng)因子的受體,是在神經(jīng)系統(tǒng)發(fā)育過程中調(diào)控神經(jīng)元存活與死亡的重要分子。正常成年人腦內(nèi)p75NTR表達(dá)水平很低且主要表達(dá)于基底前腦的膽堿能神經(jīng)元。研究發(fā)現(xiàn),p75NTR也是Aβ的受體,介導(dǎo)Aβ的神經(jīng)毒性作用,從而引起神經(jīng)軸突變性和死亡[1]。本研究小組發(fā)現(xiàn),p75NTR在AD轉(zhuǎn)基因小鼠(APPswe/PS1dE9)腦內(nèi)表達(dá)增高并參與了Aβ沉積的調(diào)控[2]。本研究探討p75NTR在AD患者腦內(nèi)的表達(dá)及其與老年斑的空間位置關(guān)系。
1.1 研究對(duì)象 雄性APPswe/PS1dE9雙轉(zhuǎn)基因小鼠來自美國(guó)Jackson動(dòng)物中心,該小鼠攜帶人致病型淀粉樣前體蛋白(amyloid precursor protein,APP)和早老素1(presenilin-1,PS1)基因,12月齡相當(dāng)于AD晚期,以同窩野生型雄性小鼠作為對(duì)照。本研究采用的AD患者(4例)和同齡認(rèn)知功能正常對(duì)照(4例)人腦標(biāo)本由美國(guó)Banner Sun Health Institute腦庫(kù)提供。本研究人體組織相關(guān)研究的臨床試驗(yàn)注冊(cè)號(hào)為ChiCTR-OCC-12001966。
1.2 實(shí)驗(yàn)取材 12月齡雄性APPswe/PS1dE9小鼠及同窩雄性野生型小鼠10只,用0.08g/kg戊巴比妥鈉深度麻醉,打開胸腔,從心尖灌注含0.5%NaNO2的磷酸鹽緩沖液100ml,取出大腦,左大腦半球液氮速凍,用含蛋白酶抑制劑的SDS制成勻漿,用于后續(xù)Western blotting檢測(cè),右大腦半球置入4%多聚甲醛固定。
1.3 免疫組織化學(xué)染色檢測(cè)p75NTR的表達(dá) 兔源多克隆抗p75NTR抗體(Ab9650)由美國(guó)New York大學(xué)M. Chao教授提供。石蠟包埋組織塊切片烤干后經(jīng)二甲苯脫水,0.5%H2O2室溫孵育10min滅活內(nèi)源性過氧化物酶,甲酸聯(lián)合EDTA進(jìn)行抗原修復(fù),30%馬血清封閉非特異性抗原,與1:500稀釋的p75NTR抗體反應(yīng),4℃過夜。洗膜3次,加入1:2000稀釋的生物素標(biāo)記的鼠抗兔二抗,室溫孵育1h,漂洗后加入辣根過氧化物酶標(biāo)記的鏈霉親和素室溫下孵育1h,洗膜3次,以DAB為底物顯色,經(jīng)蘇木精復(fù)染后進(jìn)行常規(guī)脫水、封片。Olympus顯微鏡觀察,在光強(qiáng)度和聚光器設(shè)置保持不變的條件下攝片。用Image J軟件定量分析AD患者腦內(nèi)p75NTR的表達(dá)情況。
1.4 Western blotting檢測(cè)p75NTR的表達(dá) 將腦勻漿離心后取上清,以SDS-PAGE凝膠行蛋白電泳,電泳完畢將蛋白轉(zhuǎn)至PVDF上。轉(zhuǎn)膜后在含5%脫脂奶粉的TBST中封閉1h,TBST洗滌3次,與1:1000稀釋的p75NTR抗體及β-actin抗體反應(yīng),4℃過夜。洗膜3次,加1:5000稀釋的二抗,室溫孵育1h,ECL化學(xué)發(fā)光法顯色,用Image J 軟件分析各條帶光密度值。
1.5 統(tǒng)計(jì)學(xué)處理 采用SPSS 18.0軟件進(jìn)行統(tǒng)計(jì)分析。計(jì)量數(shù)據(jù)以x±s表示,組間比較采用獨(dú)立樣本t檢驗(yàn),P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1 海馬部位神經(jīng)元與神經(jīng)纖維p75NTR的表達(dá)AD患者海馬部位可見增粗變性的p75NTR陽(yáng)性神經(jīng)纖維末梢,主要位于老年斑的中心部位(圖1A),而在認(rèn)知功能正常的對(duì)照者海馬內(nèi)未見增粗變性的神經(jīng)末梢。在APPswe/PS1dE9轉(zhuǎn)基因小鼠海馬內(nèi)也存在類似的增粗變性的神經(jīng)末梢(圖1D),而野生型小鼠海馬內(nèi)未見增粗變性的神經(jīng)末梢。AD患者和認(rèn)知正常對(duì)照者的海馬中可見深染的p75NTR陽(yáng)性神經(jīng)纖維(圖1B、E)。在12月齡APPswe/PS1dE9小鼠海馬內(nèi)可見增粗的p75NTR陽(yáng)性神經(jīng)纖維,而野生型小鼠未見陽(yáng)性神經(jīng)纖維(圖1C、F)。在AD患者和認(rèn)知正常者的海馬內(nèi)可見p75NTR陽(yáng)性的神經(jīng)元,而12月齡APPswe/PS1dE9小鼠和同齡野生型小鼠海馬內(nèi)未見p75NTR陽(yáng)性的神經(jīng)元(圖2)。
2.2 海馬組織勻漿p75NTR表達(dá)水平檢測(cè) Western blotting檢測(cè)顯示,在人和小鼠海馬組織勻漿中均存在p75NTR表達(dá),其中AD患者表達(dá)水平顯著高于認(rèn)知正常對(duì)照者,12月齡APPswe/PS1dE9小鼠表達(dá)水平顯著高于同齡野生型小鼠,表明AD患者和APPswe/PS1dE9小鼠腦內(nèi)p75NTR表達(dá)水平增高(圖2)。
p75NTR參與多種神經(jīng)營(yíng)養(yǎng)因子的信號(hào)傳導(dǎo),其生理功能取決于所結(jié)合的配體和協(xié)同受體[1,3-4]。P75NTR與各種神經(jīng)營(yíng)養(yǎng)因子及其前體結(jié)合參與了多種生理病理過程,與NGF、BDNF、神經(jīng)營(yíng)養(yǎng)因子3(neurotrophin-3,NT-3)及NT-4/5結(jié)合可以促進(jìn)神經(jīng)元的存活,而與它們的前體pro-NGF、pro-BDNF、pro-NT3相互作用可引起神經(jīng)元的凋亡[5-9]。p75NTR也是Aβ受體,在AD的發(fā)生過程中介導(dǎo)Aβ的神經(jīng)毒性作用,導(dǎo)致神經(jīng)元凋亡和神經(jīng)纖維變性[1,10]。我們前期研究發(fā)現(xiàn),p75NTR還具有促進(jìn)Aβ生成和調(diào)控Aβ沉積的作用[2]。因此p75NTR在AD發(fā)生過程中參與了Aβ生成、沉積和神經(jīng)毒性等方面的調(diào)控,是AD病理過程中的一個(gè)重要受體。
圖1 海馬部位神經(jīng)纖維p75NTR的表達(dá)及其與老年斑的位置關(guān)系(DAB ×400)Fig.1p75NTR expression on neurofibrils in hippocampus and its spatial distribution in relation to amyloid plaque (DAB ×400)
圖2 p75NTR在海馬神經(jīng)元的表達(dá)Fig.2Neuronal expression of p75NTR in hippocampus
本研究發(fā)現(xiàn),AD患者和認(rèn)知正常對(duì)照者海馬內(nèi)均存在表達(dá)p75NTR的神經(jīng)纖維,并觀察了AD患者腦內(nèi)p75NTR陽(yáng)性神經(jīng)纖維與老年斑的關(guān)系,發(fā)現(xiàn)與APPswe/PS1dE9小鼠一樣,AD患者海馬中變性的p75NTR陽(yáng)性神經(jīng)纖維也位于老年斑中心部位。這一現(xiàn)象支持我們既往在動(dòng)物模型中提出的p75NTR陽(yáng)性的變性神經(jīng)纖維可能參與了老年斑形成的假說[2]。
關(guān)于p75NTR在AD條件下的表達(dá)是增高還是降低,目前尚無定論。p75NTR主要表達(dá)于基底前腦的膽堿能神經(jīng)元及其軸突,由于這些膽堿能神經(jīng)元在AD腦內(nèi)死亡最多,因此以往許多研究認(rèn)為p75NTR在AD腦內(nèi)的表達(dá)應(yīng)該是降低的[1,11-13]。在本研究中,我們同時(shí)將AD患者和APPswe/PS1dE9小鼠腦組織與正常對(duì)照比較,結(jié)果均表明AD腦內(nèi)p75NTR表達(dá)水平顯著高于對(duì)照,表明AD條件下腦內(nèi)p75NTR的表達(dá)水平是增高的。可溶性的Aβ含量在AD起病早期增加[14],我們推測(cè)可能Aβ與p75NTR的結(jié)合抑制了p75NTR的降解,從而提高了p75NTR的水平,也可能是Aβ與p75NTR結(jié)合本身作為一種信號(hào)促進(jìn)了p75NTR自身基因的轉(zhuǎn)錄及蛋白表達(dá)。最近的研究表明,Aβ可引起人神經(jīng)母細(xì)胞瘤細(xì)胞SKSY5Y中p75NTR的表達(dá)增加[15],類胰島素生長(zhǎng)因子1受體(insulin-like growth factor 1receptor,IGF-1R)信號(hào)通路參與了Aβ對(duì)p75NTR表達(dá)的上調(diào)作用[16]。
雖然大多數(shù)證據(jù)表明Aβ是AD的核心致病物質(zhì),但在各種APP轉(zhuǎn)基因小鼠模型中,盡管腦內(nèi)出現(xiàn)Aβ過度產(chǎn)生和老年斑形成,卻并無明顯的神經(jīng)元丟失和腦萎縮[17]。對(duì)于這一現(xiàn)象目前尚無合理解釋。本研究發(fā)現(xiàn)在人(AD患者和認(rèn)知正常對(duì)照者)海馬神經(jīng)元上p75NTR表達(dá)明顯,而老齡APPswe/PS1dE9及野生型小鼠海馬神經(jīng)元未見明顯p75NTR表達(dá)。這一人與小鼠的差異現(xiàn)象對(duì)于解釋AD動(dòng)物模型和AD患者神經(jīng)元死亡及腦萎縮方面的差異具有重要的意義。由于p75NTR是Aβ的受體,介導(dǎo)Aβ引起的神經(jīng)元變性死亡,人腦神經(jīng)元表達(dá)較高水平的p75NTR,而小鼠腦內(nèi)神經(jīng)元p75NTR表達(dá)水平低,提示人腦神經(jīng)元可能比小鼠神經(jīng)元對(duì)Aβ的神經(jīng)毒性更為敏感,從而在AD患者腦內(nèi)出現(xiàn)大量神經(jīng)元死亡和腦萎縮,而過表達(dá)Aβ的轉(zhuǎn)基因AD動(dòng)物無明顯神經(jīng)元死亡和腦萎縮。
最近有研究報(bào)道體外培養(yǎng)的基底前腦神經(jīng)元中p75NTR能夠介導(dǎo)Aβ的內(nèi)吞及其在溶酶體的降解[18],提示p75NTR在Aβ的中樞清除機(jī)制中具有重要的作用。Aβ是AD的核心致病物質(zhì),p75NTR參與了Aβ的生成、沉積、降解、清除及神經(jīng)毒性作用,在AD的發(fā)生發(fā)展中具有重要作用,可能成為一個(gè)新的AD治療靶點(diǎn)。
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