徐明亮，龍志敏，廖娥，鐘國(guó)超，杜燁鴻，駱世芳，賀桂瓊

卵巢切除對(duì)APP/PS1雙轉(zhuǎn)基因小鼠腦內(nèi)老年斑及自噬的影響

徐明亮，龍志敏，廖娥，鐘國(guó)超，杜燁鴻，駱世芳，賀桂瓊

目的研究雌激素對(duì)APP/PS1雙轉(zhuǎn)基因小鼠腦內(nèi)老年斑及自噬的影響，探討雌激素缺乏導(dǎo)致阿爾茨海默病(AD)的病理機(jī)制。方法將3月齡雌性APP/PS1雙轉(zhuǎn)基因AD模型小鼠分為卵巢切除組與假手術(shù)組，術(shù)后2個(gè)月采用免疫組化染色及透射電鏡觀察兩組小鼠腦內(nèi)老年斑及自噬功能的變化，同時(shí)檢測(cè)自噬相關(guān)蛋白LC3、Beclin-1的表達(dá)情況。結(jié)果免疫組化結(jié)果顯示，與假手術(shù)組相比，卵巢切除組小鼠腦內(nèi)老年斑顯著增加，分布更廣，自噬相關(guān)蛋白LC3、Beclin-1陽(yáng)性神經(jīng)元數(shù)量均顯著下降。電鏡觀察可見(jiàn)，與假手術(shù)組相比，卵巢切除組小鼠海馬神經(jīng)元及突起腫脹明顯，暗神經(jīng)元增加，神經(jīng)元突起周圍自噬小泡顯著增多。結(jié)論雌激素缺乏可導(dǎo)致自噬泡成熟受阻、活性降低、自噬功能減弱，從而導(dǎo)致AD病理改變加重。

卵巢切除術(shù)；阿爾茨海默病；老年斑；自噬；小鼠，轉(zhuǎn)基因

阿爾茨海默病(Alzheimer disease，AD)是一種發(fā)病機(jī)制未明的慢性進(jìn)行性神經(jīng)系統(tǒng)變性疾病[1]，是臨床上最為常見(jiàn)的癡呆類型。神經(jīng)細(xì)胞外淀粉樣β蛋白(amyloid β peptide，Aβ)沉積形成的老年斑是AD的典型病理特征之一[2]。研究顯示，女性絕經(jīng)后罹患AD的風(fēng)險(xiǎn)較同齡男性更高，這與絕經(jīng)后女性腦內(nèi)雌激素水平驟降及隨后持續(xù)處于低水平有關(guān)[3-4]，但雌激素缺乏導(dǎo)致AD的分子機(jī)制尚不明確。研究表明，細(xì)胞自噬異常在AD發(fā)病中起重要作用，自噬泡被認(rèn)為是Aβ產(chǎn)生的源頭[5]。那么，雌激素缺乏是否影響自噬泡的數(shù)量或活性，進(jìn)而影響體內(nèi)Aβ的水平及老年斑的形成？關(guān)于這一問(wèn)題尚無(wú)文獻(xiàn)報(bào)道。本課題擬以卵巢切除AD模型小鼠為研究對(duì)象，應(yīng)用形態(tài)學(xué)檢測(cè)手段，觀察雌激素缺乏對(duì)癡呆小鼠腦內(nèi)老年斑形成及自噬功能的影響，以探討AD發(fā)生發(fā)展的病理機(jī)制。

1 材料與方法

1.1 動(dòng)物及試劑 APP/PS1雙轉(zhuǎn)基因癡呆模型小鼠購(gòu)于中國(guó)醫(yī)學(xué)科學(xué)院醫(yī)學(xué)實(shí)驗(yàn)動(dòng)物研究所，體重25～35g，進(jìn)行基因鑒定后喂養(yǎng)于重慶醫(yī)科大學(xué)動(dòng)物中心SPF級(jí)動(dòng)物房。實(shí)驗(yàn)遵循實(shí)驗(yàn)動(dòng)物使用倫理相關(guān)要求。鼠抗4G8單克隆抗體購(gòu)自Convance公司，兔抗LC3多克隆抗體購(gòu)自Sigma公司，兔抗Beclin-1多克隆抗體購(gòu)自Abcam公司，小鼠二步法免疫組化試劑盒、兔二步法免疫組化試劑盒、DAB試劑盒均購(gòu)自北京中杉金橋生物技術(shù)有限公司，其他試劑均為國(guó)產(chǎn)分析純。

1.2 實(shí)驗(yàn)方法

1.2.1 動(dòng)物模型建立 3月齡APP/PS1雙轉(zhuǎn)基因雌性小鼠共16只，隨機(jī)分為假手術(shù)組與卵巢切除組(n=8)。經(jīng)3.5%水合氯醛麻醉后暴露腹腔，切除雙側(cè)卵巢，制成雌激素缺乏模型。假手術(shù)組麻醉后暴露腹腔，不做其他處理，逐層縫合后2組小鼠均于SPF級(jí)條件中持續(xù)喂養(yǎng)2個(gè)月。

1.2.2 標(biāo)本制備 小鼠麻醉后，開(kāi)胸暴露心臟。經(jīng)左心室插管至升主動(dòng)脈，剪開(kāi)右心耳，灌注生理鹽水，冰上斷頭取腦。將腦組織置于4%多聚甲醛溶液中固定，常規(guī)石蠟包埋，冠狀切片，厚度4μm。

1.2.3 形態(tài)學(xué)觀察 免疫組化染色：石蠟切片經(jīng)二甲苯脫蠟，梯度乙醇水化，PBS清洗5min×3次，枸櫞酸鈉微波抗原修復(fù)，室溫自然冷卻，PBS清洗5min×3次，3%H2O210min消除內(nèi)源性過(guò)氧化物酶，PBS沖洗5min×3次，加入一抗(抗體稀釋比例4G8為1:500，LC3為1:400，Beclin-1為1:400)，4℃冰箱過(guò)夜。次日用PBS清洗5min×3次，加入相應(yīng)二抗，室溫孵育60min，PBS清洗5min×3次。加入DAB顯色，顯微鏡下觀察并適時(shí)終止顯色，梯度乙醇和二甲苯1、二甲苯2脫水透明后用中性樹(shù)膠封固，光鏡下觀察并采集圖像。計(jì)數(shù)4G8陽(yáng)性斑塊數(shù)量并計(jì)算其面積，計(jì)數(shù)Beclin-1、LC3陽(yáng)性神經(jīng)元數(shù)量。透射電鏡觀察：小鼠用4%多聚甲醛、2.5%戊二醛灌注固定后，在解剖鏡下取小鼠海馬CA1區(qū)1mm3組織塊，2.5%戊二醛中固定2h，1%四氧化鋨固定，乙醇丙酮梯度脫水，環(huán)氧樹(shù)脂包埋聚合，半薄切片定位，超薄切片，鉛鈾染色，透射電鏡(Hitachi-7500)下觀察腦組織超微結(jié)構(gòu)。

1.3 統(tǒng)計(jì)學(xué)處理 采用SPSS 17.0軟件進(jìn)行數(shù)據(jù)處理。計(jì)量資料以表示，組間比較采用兩樣本均數(shù)的t檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 卵巢切除后小鼠腦皮質(zhì)及海馬Aβ沉積的變化

免疫組化染色顯示，兩組AD小鼠腦內(nèi)均出現(xiàn)了4G8陽(yáng)性斑塊，說(shuō)明兩組小鼠腦組織神經(jīng)元外均有Aβ的沉積。但假手術(shù)組小鼠腦內(nèi)的散在陽(yáng)性斑塊主要分布在大腦皮質(zhì)，海馬內(nèi)偶見(jiàn)。卵巢切除小鼠大腦皮質(zhì)和海馬內(nèi)4G8陽(yáng)性斑塊數(shù)量明顯增多(P=0.001)、斑塊面積增加(P=0.011，圖1)。透射電鏡觀察顯示：兩組小鼠海馬內(nèi)均出現(xiàn)神經(jīng)元突起腫脹。與假手術(shù)組相比，卵巢切除組小鼠海馬內(nèi)暗神經(jīng)元增多，且神經(jīng)元突起周圍出現(xiàn)數(shù)量較多的自噬小泡(圖2)。

圖1 兩組小鼠腦內(nèi)老年斑情況比較(免疫組化染色×400)Fig.1 Senile plaques (SP) in the brain of two groups of shamoperated AD (Sham-AD) and ovariectomy AD (OVX-AD) mice (Immunohistochemistry staining ×400)

2.2 卵巢切除后小鼠腦內(nèi)自噬相關(guān)蛋白表達(dá)的變化 免疫組化染色顯示，兩組小鼠腦內(nèi)均出現(xiàn)較多的自噬相關(guān)蛋白Beclin-1陽(yáng)性神經(jīng)元，假手術(shù)組小鼠腦內(nèi)Beclin-1陽(yáng)性神經(jīng)元數(shù)量較多(88.50±12.45個(gè))，但免疫陽(yáng)性物主要分布于近胞膜胞質(zhì)處，卵巢切除組小鼠腦內(nèi)的Beclin-1陽(yáng)性神經(jīng)元數(shù)目明顯減少(44.00±3.74，P<0.001)且免疫陽(yáng)性物均勻分布于整個(gè)胞質(zhì)。另一自噬相關(guān)蛋白LC3免疫陽(yáng)性細(xì)胞也廣泛分布于兩組小鼠腦內(nèi)，假手術(shù)組小鼠腦內(nèi)LC3陽(yáng)性神經(jīng)元數(shù)量較多(88.50±12.45個(gè))，胞體和突起濃染，且突起較長(zhǎng)，而卵巢切除組小鼠腦內(nèi)LC3陽(yáng)性神經(jīng)元數(shù)量明顯減少(62.00±11.92個(gè)，P=0.006)，雖然胞體和突起均濃染，但突起較短，提示突起可能有變性、斷裂(圖3)。

圖2 兩組小鼠腦組織超微結(jié)構(gòu)的電鏡觀察(×4000)Fig.2 Ultrastructures of neurons under transmission electron microscopy (TEM) in the 2 groups (×4000)

圖3 兩組小鼠大腦皮質(zhì)Beclin-1與LC3表達(dá)情況(免疫組化 ×400)Fig.3 Expression of Beclin-1 and LC3 in the brain of two groups of sham-operated AD and ovariectomy AD (OVX-AD) mice (Immunohistochemistry staining ×400)

3 討 論

老年斑(senile plaque，SP)是AD腦部典型的病理特征之一[6]，主要由Aβ聚集和沉積而成，Aβ被認(rèn)為是AD發(fā)病的始動(dòng)因素[7-9]。Aβ由淀粉蛋白前體蛋白(amyloid precursor protein，APP)經(jīng)β-分泌酶和早老素1(presenilin 1，PS1)/γ-分泌酶相繼切割產(chǎn)生[10]。研究已證實(shí)APP與PS的基因突變是引起家族性AD的主要原因[11-12]。本實(shí)驗(yàn)所用小鼠同時(shí)攜帶“瑞典家族”突變的APP(APPswe)與PS1(PS△E9)基因：APPswe基因中，突變發(fā)生在APP編碼序列末端的670(Lvs/Asn)、671(Met/Leu)位點(diǎn)，PS△E9則是在家族性AD中發(fā)現(xiàn)的第9個(gè)外顯子缺失突變。此類小鼠隨月齡增加出現(xiàn)認(rèn)知功能的進(jìn)行性損害，腦組織出現(xiàn)SP沉積及神經(jīng)元丟失，類似AD患者的病理特征。因此，APP/PS1雙轉(zhuǎn)基因小鼠被認(rèn)為是研究AD病理機(jī)制及藥物篩選的理想動(dòng)物模型[13]。

本研究對(duì)3月齡APP/PS1小鼠行雙側(cè)卵巢切除，術(shù)后2個(gè)月觀察到小鼠腦內(nèi)SP數(shù)量增加，體積增大，且有大量小體積的新生SP產(chǎn)生，同時(shí)SP的分布范圍更廣，可見(jiàn)于大腦皮質(zhì)和海馬等腦區(qū)。電鏡觀察還發(fā)現(xiàn)卵巢切除小鼠腦內(nèi)神經(jīng)元突起腫脹明顯，暗神經(jīng)元數(shù)量增多，此外，Aβ沉積及神經(jīng)元變性等病理特征有加重趨勢(shì)，提示雌激素缺乏可加速AD的病程。本研究結(jié)果與AD流行病學(xué)調(diào)查相吻合。流行病學(xué)調(diào)查顯示，絕經(jīng)后婦女AD的發(fā)病率比同年齡男性高1.5～3倍，女性AD患者腦內(nèi)的病理改變及認(rèn)知功能障礙較男性患者嚴(yán)重，這可能與絕經(jīng)后雌激素水平下降有關(guān)[3]。因此，本研究采用的雙側(cè)卵巢切除術(shù)AD模型小鼠可模擬臨床絕經(jīng)期女性AD患者，是探尋雌激素缺乏導(dǎo)致AD發(fā)生發(fā)展病理機(jī)制的一種可靠動(dòng)物模型[14]。

本研究觀察到，卵巢切除AD模型小鼠腦內(nèi)自噬小泡顯著增多，且主要圍繞在軸突周圍，提示雌激素缺乏可誘導(dǎo)自噬泡增加。自噬是細(xì)胞利用溶酶體降解自身受損的細(xì)胞器和錯(cuò)誤折疊的蛋白質(zhì)等有害大分子物質(zhì)的重要途徑[15]，是真核細(xì)胞特有的生命現(xiàn)象[16]。細(xì)胞接受自噬誘導(dǎo)信號(hào)后，首先形成雙層扁平狀膜結(jié)構(gòu)的吞噬泡，并逐漸擴(kuò)展包裹待降解物質(zhì)，兩端融合形成密閉球狀的自噬體。自噬體沿微管運(yùn)行，與多泡體或內(nèi)涵體融合并成熟。成熟的自噬體與溶酶體融合形成單層膜的自噬溶酶體，進(jìn)而酸化，激活酸性蛋白水解酶執(zhí)行降解功能[17]。自噬途徑異常表現(xiàn)為早期自噬體在數(shù)量和體積上的增加。細(xì)胞自噬異常在AD發(fā)病中發(fā)揮重要作用[5,18-19]，研究發(fā)現(xiàn)自噬泡(包括自噬體和自噬溶酶體)中富含APP、Aβ及γ-分泌酶，誘導(dǎo)自噬可引起Aβ增多，因此，自噬泡被認(rèn)為是Aβ產(chǎn)生的場(chǎng)所[5,20]。AD患者及模型鼠腦中各種不成熟自噬泡的異常增多是自噬被激活的有力證據(jù)[21]。本實(shí)驗(yàn)觀察到卵巢切除小鼠腦內(nèi)Aβ沉積加重，可能系雌激素缺乏導(dǎo)致自噬小泡增多所致。

有研究表明，自噬具有神經(jīng)保護(hù)作用，中樞神經(jīng)系統(tǒng)基礎(chǔ)水平的自噬受抑制可導(dǎo)致泛素化蛋白的累積和神經(jīng)退行性疾病，誘導(dǎo)自噬可加速有毒物質(zhì)的清除[22-23]。本研究觀察到卵巢切除后自噬泡顯著增加，但增加的自噬泡不僅未能清除具有神經(jīng)毒性的Aβ，反而導(dǎo)致Aβ沉積增加，所以我們推測(cè)雌激素缺乏可能導(dǎo)致了自噬成熟受阻、自噬活性降低。為驗(yàn)證此假設(shè)，本研究觀察了反映自噬活性的分子標(biāo)志物Beclin-1，發(fā)現(xiàn)卵巢切除小鼠腦內(nèi)Beclin-1表達(dá)顯著低于對(duì)照組小鼠，且另一自噬相關(guān)蛋白LC3的表達(dá)改變與其一致。上述結(jié)果表明雌激素缺乏可影響自噬溶酶體的成熟，從而導(dǎo)致自噬活性降低、自噬功能減弱，相應(yīng)地，不成熟自噬泡聚集增加，Aβ降解受阻，從而導(dǎo)致Aβ的沉積增多。但雌激素缺乏導(dǎo)致自噬異常的機(jī)制尚有待深入研究。

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Effects of ovariectomy on autophagy and senile plaques in the brain of adult APP/PS1 double transgenic mice

XU Ming-liang1, LONG Zhi-min1, LIAO E1, ZHONG Guo-chao1, DU Ye-hong1, LUO Shi-fang1,2, HE Gui-qiong1,2*1Institute of Neuroscience,2Department of Human Anatomy, Chongqing Medical University, Chongqing 400016, China

*Corresponding author, E-mail: guiqionghe@hotmail.com

This work was supported by the National Natural Science Foundation of China (81371221), the Program for New Century Excellent Talents in University of the National Department of Education (NCET-11-1084), the Technology Foundation for Selected Overseas Chinese Scholar, Ministry or Personnel of China[(2011)235], and the College Students' Scientific Research and Innovation Experimental Program of Chongqing Medical University (201221)

ObjectiveTo investigate the effect of ovarian estrogen on autophagy and senile plaques (SP) in the brain of adult APP/PS1 double transgenic mice, and inquire into the mechanism of Alzheimer disease (AD) neuropathogenesis due to estrogen deficiency.MethodsAPP/PS1 AD mice (3-month old) were divided into ovariectomy group (OVX-AD) and sham group (sham-AD). Two months after operation, transmission electron microscopy (TEM) was employed to observe the morphological structures of the brains of AD model mice, and immunohistochemical staining was performed to examine the changes in SP and autophagy-related proteins LC3 and Beclin-1 in the brain of AD model mice.ResultsImmunohistochemical staining showed that the number of SP in the brain of OVX-AD mice incrcased significantly compared with that in sham-AD mice, and distribution of SP was observed more extensively, including the cortex and hippocampus. The autophagy related proteins LC3 and Beclin-1 positive neurons were decreased greatly in the OVX-AD mice brain compared with that of sham-AD mice. TEM revealed a larger number of swollen and dark neurons in hippocampus of OVX-AD mice, while more autophagosome was observed around the neuronal processes in OVX-AD mice brain compared with sham-AD mice. Conclusion Estrogen deficiency may lead to delayed maturation of autophagy, decrease the activity and weaken the function of autophagy, which in turn may result in aggravation of pathological features of AD.

ovariectomy; Alzheimer disease; senile plaques; autophagy; mice, transgenic

R749.16

A

0577-7402(2015)01-0026-04

10.11855/j.issn.0577-7402.2015.01.06

2014-05-08；

2014-11-20)

(責(zé)任編輯：沈?qū)?

國(guó)家自然科學(xué)基金(81371221)；教育部“新世紀(jì)優(yōu)秀人才”支持計(jì)劃(NCET-11-1084)；留學(xué)人員科技活動(dòng)項(xiàng)目擇優(yōu)資助[渝人社辦(2011)235號(hào)]；重慶醫(yī)科大學(xué)大學(xué)生科研與創(chuàng)新實(shí)驗(yàn)項(xiàng)目(201221)

徐明亮，碩士研究生。主要從事阿爾茨海默病發(fā)病機(jī)制及治療方面的研究

400016 重慶 重慶醫(yī)科大學(xué)神經(jīng)科學(xué)研究中心(徐明亮、龍志敏、廖娥、鐘國(guó)超、杜燁鴻)，解剖學(xué)教研室(駱世芳、賀桂瓊)

]賀桂瓊，E-mail：guiqionghe@hotmail.com