胡純純 劉春雪 徐 秀

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·綜述·

孤獨(dú)癥譜系障礙動(dòng)物模型的研究進(jìn)展

胡純純 劉春雪 徐 秀

孤獨(dú)癥譜系障礙(ASD)是一種神經(jīng)發(fā)育障礙性疾病[1]，其核心癥狀為社交和(或)溝通障礙，狹隘興趣與刻板行為。目前，ASD的病因尚不明確。多數(shù)研究認(rèn)為，遺傳[2]與發(fā)育的早期環(huán)境因素[3]共同作用，在ASD的病因中發(fā)揮了關(guān)鍵作用。

世界大范圍的流行病學(xué)調(diào)查中，ASD在人群中的發(fā)病率約為1%，男性是女性的2～3倍[4,5]。美國(guó)近期流行病學(xué)調(diào)查ASD的發(fā)病率達(dá)到1/68[6]。因此，深入了解ASD的病因和機(jī)制，尋求治療顯得尤為重要。目前，神經(jīng)生物學(xué)研究已經(jīng)明確了ASD的腦血流灌注模式和神經(jīng)生化特點(diǎn)。此外，在ASD的神經(jīng)系統(tǒng)連接特點(diǎn)、神經(jīng)解剖和細(xì)胞與分子水平上也取得了一些進(jìn)展[7]。然而，以ASD患兒為樣本的機(jī)制研究仍舊存在很多的局限性，①通常不能獲得高質(zhì)量的尸檢腦組織資源，②神經(jīng)影像學(xué)所能提供的信息非常有限，③各種基因型與表型的臨床資料非常零散等。因此，構(gòu)筑動(dòng)物模型可以更好地了解ASD的神經(jīng)分子生物特性與腦解剖特點(diǎn)，以及明確各類不同的認(rèn)知與行為亞型。

1 ASD動(dòng)物模型

動(dòng)物模型作為研究ASD的一個(gè)工具，其優(yōu)勢(shì)在于：①取得在活體中難以取得的神經(jīng)組織標(biāo)本；②便于控制變量，研究基因功能；③觀察生化或基因在ASD神經(jīng)病理學(xué)中的作用；④觀察社會(huì)行為；⑤獲得干預(yù)療效等。動(dòng)物模型也存在局限性:①無法完全模擬人類的病理生理機(jī)制，②動(dòng)物社交行為與人存在差異等。

一個(gè)理想的動(dòng)物模型需具備3種效度，①表面效度：需包含疾病的典型表現(xiàn)；②構(gòu)建效度：需與疾病的生物學(xué)機(jī)制類似；③預(yù)測(cè)效度：對(duì)疾病的有效干預(yù)能在模型中產(chǎn)生預(yù)期結(jié)果[8,9]?，F(xiàn)已用于ASD研究的動(dòng)物模型有果蠅、斑馬魚、鼠和靈長(zhǎng)類動(dòng)物等，本文就生化作用誘導(dǎo)、基因分子缺陷和以生化作用為基礎(chǔ)的基因型建立的ASD動(dòng)物模型進(jìn)行文獻(xiàn)復(fù)習(xí)和歸納。

2 生化誘導(dǎo)ASD動(dòng)物模型

近年來的研究表明，環(huán)境因素在ASD病因?qū)W中起著重要作用[10～12]。而胎兒發(fā)育關(guān)鍵期(如胎兒期或圍生期)暴露于環(huán)境致病因素，致孕期感染或炎癥反應(yīng)而導(dǎo)致ASD，也是其病因研究的重點(diǎn)[13,14]。通過動(dòng)物模型，能夠得到生化因素誘導(dǎo)后其腦解剖與行為異常的結(jié)果，為研究神經(jīng)系統(tǒng)功能、篩選新藥和探索治療方法提供新的思路。

VPA鼠模型是經(jīng)典的ASD動(dòng)物模型，而近期實(shí)驗(yàn)研究則更注重VPA劑量及時(shí)間差異效應(yīng)下的不同損害。

VPA的劑量研究實(shí)驗(yàn)發(fā)現(xiàn)[18～21]，孕期第12.5 d單次腹腔注射VPA 600 mg·kg-1可以誘發(fā)子鼠典型的ASD表現(xiàn)。此動(dòng)物模型除有ASD的核心癥狀外，對(duì)疼痛敏感性降低而對(duì)非疼痛刺激敏感性增高。Banerjee等[22]通過對(duì)12.5 d的孕期母鼠注射不同劑量的VPA(500, 600 mg·kg-1),并運(yùn)用條件恐懼裝置和社交互動(dòng)裝置等測(cè)試子鼠，發(fā)現(xiàn)雖然劑量相似，但注射600 mg子鼠模型有更明顯的焦慮行為，在社交互動(dòng)區(qū)域花費(fèi)的時(shí)間更少，并且在社交情緒學(xué)習(xí)方面存在異常，這為ASD社交情緒學(xué)習(xí)的研究提供了方向。

Reynolds等[23]在VPA時(shí)間差異效應(yīng)研究中發(fā)現(xiàn)，在幼鼠出生后6～12 d注射VPA 150 mg·kg-1·d-1，此ASD幼鼠的聽覺敏感性低，與既往孕期母鼠注射VPA的ASD子鼠模型聽覺敏感性高不同，說明在大腦發(fā)育的不同階段暴露于VPA可能造成的不同損害。

而VPA對(duì)其他動(dòng)物模型的影響，Lee等[24]也做過有趣的實(shí)驗(yàn)。將受精5 d后的斑馬仔魚放置于含VPA 2 mmol·L-1的魚缸中急性處理3 h，與對(duì)照組比較，發(fā)現(xiàn)斑馬魚在發(fā)育過程暴露于VPA后出現(xiàn)短暫的部分神經(jīng)細(xì)胞增殖減少，但未在實(shí)驗(yàn)組成魚后觀察到與其他動(dòng)物類似的行為缺陷。這種情況應(yīng)與斑馬魚在發(fā)育過程中神經(jīng)修復(fù)有關(guān)。Jacob

等[25]將斑馬魚的受精胚胎去卵膜后，分別在3～24 h、～48 h和～72 h用VPA 0.625 mmol·L-1處理，發(fā)現(xiàn)斑馬魚5-HT神經(jīng)元分化受損，使其產(chǎn)生ASD樣癥狀，并且在～48 h的處理效果最為明顯，而在～72 h后則對(duì)5-HT的表達(dá)無影響。這些神經(jīng)分子機(jī)制的研究將對(duì)未來分子靶向治療提供重要的線索。

2.2 丙酸(PPA)動(dòng)物模型 PPA是一種短鏈的脂肪酸，作為腸道細(xì)菌的代謝終產(chǎn)物，常用作食品防腐劑。文獻(xiàn)報(bào)道存在胃腸紊亂的ASD兒童其短鏈脂肪酸等生物標(biāo)志物存在異常[26]，并伴免疫異常[27,28]。

PPA鼠模型分別從免疫生化及行為學(xué)方面的改變證明了PPA與ASD的相關(guān)性。Shultz等[29]報(bào)道鼠腦室內(nèi)注射PPA可導(dǎo)致解剖和行為方面的異常，其腦室出現(xiàn)反應(yīng)性角質(zhì)化與活化的小膠質(zhì)細(xì)胞，并通過行為學(xué)實(shí)驗(yàn)，發(fā)現(xiàn)PPA鼠存在學(xué)習(xí)記憶障礙，刻板重復(fù)行為及感覺運(yùn)動(dòng)異常，類似于人類ASD。MacFabe等[30]也進(jìn)一步證實(shí)了前者的實(shí)驗(yàn)，連續(xù)3 d將PPA 250 mg·kg-1注射入雄性成鼠中，并對(duì)其腦組織做免疫組化分析，發(fā)現(xiàn)腦組織中膠質(zhì)化反應(yīng)性星形細(xì)胞和被激活的小膠質(zhì)細(xì)胞，揭示了其最初的神經(jīng)免疫性反應(yīng)。El-Ansary等[31]將引起神經(jīng)毒性的PPA劑量(250 mg·kg-1)注入成年白化鼠中，發(fā)現(xiàn)社交行為障礙、典型的認(rèn)知功能損害和重復(fù)行為，并且伴隨氧化應(yīng)激標(biāo)志物的上升和谷胱甘肽的下降，其谷胱甘肽過氧化物酶和過氧化氫酶的活性也隨之降低。Foley等[27]對(duì)孕12～16 d的胎鼠每日注射PPA 500 mg·kg-1，與對(duì)照組比較，其尋巢反應(yīng)能力減弱，提示在嗅覺誘導(dǎo)的社交認(rèn)知中存在障礙，并且青春期的雄鼠相比于對(duì)照組有更多的探索行為，提示社交記憶的缺陷。因此，有研究者提出，在胎兒期PPA暴露可對(duì)新生鼠、青春期鼠和成年鼠社交行為產(chǎn)生一定的影響。PPA導(dǎo)致的神經(jīng)毒性也許能作為一個(gè)重要的環(huán)境因素成為ASD的病因。

2.3 雙酚基丙烷(BPA)動(dòng)物模型 BPA是重要的有機(jī)化工原料，是苯酚和丙酮的重要衍生物。被廣泛用于罐頭食品和飲料的包裝等。作為內(nèi)分泌干擾物，胎兒期及圍生期的暴露將導(dǎo)致兒童神經(jīng)發(fā)育的異常[32,33]及行為的改變[34～36]。很多研究都揭示了內(nèi)分泌干擾素與ASD及注意力缺陷多動(dòng)障礙的關(guān)系[37]。

Wolstenholme等[34]對(duì)孕期暴露BPA后的連續(xù)3代鼠行社會(huì)認(rèn)知測(cè)試，與對(duì)照組比較，表現(xiàn)出更多的探索行為，不僅如此，3代鼠缺乏對(duì)新刺激(陌生鼠)的認(rèn)知，說明在社交記憶中存在缺陷；而在曠場(chǎng)試驗(yàn)中則表現(xiàn)過度活躍。證實(shí)了BPA對(duì)鼠的社會(huì)認(rèn)知及行為等方面具有長(zhǎng)期的影響。

而BPA對(duì)其它動(dòng)物模型也存在影響。Kaurr等[38]運(yùn)用黑腹果蠅作為BPA動(dòng)物模型。給果蠅喂食不同劑量的BPA(0、0.1、0.5、1 mmol·L-1)食物，觀察果蠅在自主運(yùn)動(dòng)方面的差異，發(fā)現(xiàn)其較對(duì)照組有更多的重復(fù)行為(如梳理行為)，且有非正常的社交互動(dòng)(與周圍果蠅距離縮短)，而喂食0.5 mmol·L-1的果蠅癥狀最具典型性。這些結(jié)論也證明了果蠅作為神經(jīng)發(fā)育研究動(dòng)物模型的可行性，均存在ASD相關(guān)行為。

2.4 其它生化模型 經(jīng)過數(shù)十年的研究，各個(gè)學(xué)者均提出過不同的生化模型假說。比如抗體模型[39,40]，通過誘導(dǎo)鼠模型的研究，證實(shí)了母體產(chǎn)生的特異性抗體(IgE13-IgE18及IgG)，能夠影響胎兒的神經(jīng)發(fā)育而產(chǎn)生ASD樣行為[41];博爾納病毒(BDV)模型[42],BDV感染后的成鼠表現(xiàn)出更多的社交障礙及刻板行為，類似于ASD患兒[43];氯蜱硫磷模型，雖未能證明對(duì)人類產(chǎn)生ASD樣癥狀[44]，但在斑馬魚暴露于大劑量氯蜱硫毒藥的研究中[45]，則證實(shí)能誘導(dǎo)其產(chǎn)生ASD樣行為；過量高頻射線模型[46]，將恒河短尾猿在孕早期暴露于過度的X射線中，發(fā)現(xiàn)成年猿相較于對(duì)照組有重復(fù)刻板行為，重復(fù)語(yǔ)言及認(rèn)知行為損害，而臨床研究證實(shí)，高頻射線能導(dǎo)致ASD發(fā)病率升高[47]。在人和動(dòng)物模型的研究中，發(fā)現(xiàn)環(huán)境因素均占有非常重要的作用，因此，不同的生化模型對(duì)ASD的研究均具有非常重要的意義。表1總結(jié)生化誘導(dǎo)劑致鼠ASD表型及神經(jīng)生化改變。

表1 生化誘導(dǎo)劑致鼠ASD表型及神經(jīng)生化改變

注 PMID 為PubMed indexed唯一標(biāo)識(shí)碼；+: 陽(yáng)性，-: 陰性;1)注射VPA的時(shí)間差異性，其鼠的聽覺敏感性出現(xiàn)升高和降低；2)PPA效應(yīng)存在性別差異，母鼠聽覺敏感性升高而雄鼠降低

3 基因誘導(dǎo)ASD動(dòng)物模型

近年來依靠基因組學(xué)研究的發(fā)展，ASD的遺傳學(xué)研究取得了巨大進(jìn)展。通過不同的基因測(cè)序技術(shù)，鑒定了大批ASD的易感及致病基因位點(diǎn)。建立于基因突變之上，能夠獲得大量的ASD動(dòng)物模型。目前熱門的動(dòng)物模型突變基因包括Fmr1、Nlgn3、Shank3、Mecp2、Ube3a、Chd8、Foxp2、Kctd13和Cntnap2等。然而，ASD的遺傳異質(zhì)性是研究機(jī)制及治療的主要瓶頸，這也為動(dòng)物模型的研究提供了一個(gè)重點(diǎn)的方向。

3.1SHANK3模型 人類基因組中有三組SHANK基因，分別為SHANK1、SHANK2和SHANK3，表達(dá)在大腦的不同區(qū)域，并在興奮性突觸后膜致密區(qū)中編碼突觸支架蛋白。在部分ASD患者中，已證實(shí)檢測(cè)到SHANK基因的突變[48]。最近，SHANK3基因成為研究ASD基因的熱點(diǎn)。

Bangash等[49]構(gòu)建的ASD鼠模型類似人類的SHANK3微缺失，即缺失羧基段。Shank3微缺失鼠表現(xiàn)為NMDA受體功能下降，使其神經(jīng)發(fā)育受損及影響突觸的可塑性，使其學(xué)習(xí)記憶受損。Wang等[50]構(gòu)建的Shank3缺陷鼠模型，通過分類Shank3相關(guān)的各個(gè)亞型，來評(píng)估Shank3的功能。不同外顯子缺失的鼠可導(dǎo)致不同程度的功能缺陷，比如自我傷害、重復(fù)梳毛和社交障礙等[50～54]。通過分類Shank3的不同基因缺失對(duì)小鼠表型的影響，可以闡述其ASD發(fā)生發(fā)展的分子機(jī)制，以期為分子靶向治療提供新思路。

除了鼠模型外，Gauthier等[55]在下調(diào)Shank3同源基因的斑馬魚模型中發(fā)現(xiàn)，與對(duì)照組相比，表現(xiàn)為頭圍的減小、游動(dòng)時(shí)對(duì)觸碰的反應(yīng)下降等。由此證明了斑馬魚的Shank3基因在其中樞神經(jīng)系統(tǒng)功能中所起的作用。

3.2FMR1模型 FMRP(Fragile X mental retardation protein)是脆性X相關(guān)蛋白(FXRP)的成員之一。其脆性X綜合征的發(fā)病被證明是由X染色體上的FMR1基因缺陷導(dǎo)致蛋白合成減少所致。部分破壞其分子通路的兒童有典型的ASD表現(xiàn)[56]。脆性X綜合征兒童的其他表現(xiàn)有認(rèn)知障礙、特殊面容、多動(dòng)和焦慮等[57～59]。

對(duì)于Fmr1缺陷的鼠模型，Hamilton等[60]將敲除Fmr1的鼠進(jìn)行了認(rèn)知、社交、學(xué)習(xí)能力和自主運(yùn)動(dòng)測(cè)試，發(fā)現(xiàn)Fmr1敲除鼠存在與人類ASD患者相似的行為學(xué)表現(xiàn)，而其它研究也證明了此結(jié)果[61～63]。

果蠅Fmr1基因與人在其蛋白功能區(qū)域的同源性為35%，相似性為56%[64,65]。研究發(fā)現(xiàn)[66,67]，F(xiàn)mr1突變果蠅存在行為學(xué)異常。Kanellopoulos等[68]發(fā)現(xiàn)果蠅dFmr下降后其突觸的mGluR水平上升，而使cAMP減少，可導(dǎo)致其聯(lián)合性的學(xué)習(xí)和記憶缺陷，從而闡述了Fmr1基因缺陷導(dǎo)致ASD樣反應(yīng)的分子機(jī)制。

3.3NLGN3模型NLGN基因編碼的蛋白是后突觸的細(xì)胞黏附蛋白成員之一。它的功能是調(diào)控海馬神經(jīng)元的形成，以及谷氨酸能后突觸蛋白的形成，并與ASD相關(guān)[69]。

Nlgn突變鼠模型在感官行為和感覺加工方面存在缺陷，并且行為方面的缺陷與ASD患者的特征有著驚人的一致性[39]。Radyushkin等[70]報(bào)道敲除Nlgn3的鼠模型在腦的不同結(jié)構(gòu)上存在差異：在灰質(zhì)結(jié)構(gòu)，如海馬，紋狀體，丘腦較正常個(gè)體為小。這種突變體的表型在人類的ASD中也有發(fā)現(xiàn)。

基因誘導(dǎo)ASD動(dòng)物模型總結(jié)見表2。

4 以生化作用為基礎(chǔ)的基因型ASD動(dòng)物模型

基因與環(huán)境的相互作用已成為ASD研究的熱點(diǎn)[85,86]。部分研究認(rèn)為，環(huán)境造成基因表達(dá)差異的差異，是最終導(dǎo)致動(dòng)物模型產(chǎn)生ASD表型的原因。Jacob等[25]發(fā)現(xiàn)由于VPA導(dǎo)致斑馬魚模型ascl1b/Ascl1基因表達(dá)的下降，從而導(dǎo)致血清胺能分化的下降。同樣，Kolozsi等[87]的實(shí)驗(yàn)將VPA與Nlgn3相結(jié)合，發(fā)現(xiàn)胎兒期暴露VPA后的小鼠Nlgn3 mRNA的表達(dá)明顯低于對(duì)照組，并認(rèn)為VPA小鼠的ASD樣行為亞型可能由于Nlgn3的低表達(dá)導(dǎo)致。這些實(shí)驗(yàn)均證明了環(huán)境與基因相互作用下的表觀遺傳學(xué)機(jī)制在疾病發(fā)生發(fā)展中的作用。

5 結(jié)論

動(dòng)物模型對(duì)于研究ASD至關(guān)重要。生化誘導(dǎo)ASD動(dòng)物模型，能夠控制變量，探索不同的環(huán)境毒素(生化因素)在ASD發(fā)病中的作用。通過生化作用誘導(dǎo)動(dòng)物模型，還可研究環(huán)境影響下神經(jīng)發(fā)育特點(diǎn)和環(huán)境生化因素影響下的表觀遺傳機(jī)制。當(dāng)動(dòng)物模型在去除環(huán)境毒素(生化)的條件后出現(xiàn)癥狀的可逆性，為研究ASD的治療提供了更多的方向?；蚍肿尤毕軦SD動(dòng)物模型，其優(yōu)勢(shì)在于目標(biāo)明確，可通過誘導(dǎo)單一基因分子缺陷來研究ASD。由于ASD基因分子缺陷的臨床資料較為零散，通過復(fù)制大量動(dòng)物樣本，能夠提高可信度。通過動(dòng)物模型，能將ASD基因型與不同表型關(guān)聯(lián)性進(jìn)行總結(jié)，以探究不同基因型的最佳干預(yù)療效。

目前，ASD運(yùn)用的動(dòng)物模型種類多種多樣 。 不同種類動(dòng)物模型也存在其各自的優(yōu)缺點(diǎn) 。 果蠅模型， 其繁殖周期短、多產(chǎn)、經(jīng)濟(jì)環(huán)保和基因較簡(jiǎn)單，但它與人類親緣關(guān)系較遠(yuǎn)[38]。斑馬魚模型，作為新興模型，其具有繁殖快、交配行為受光周期控制、產(chǎn)卵量多、受精卵在體外受精、前期胚胎整體透明、便于使用藥物和易于飼養(yǎng)等優(yōu)勢(shì)[84]。鼠模型是ASD研究的經(jīng)典模型，其基因型與人同源性較高，較為經(jīng)濟(jì)，而對(duì)其行為觀察及學(xué)習(xí)記憶的技術(shù)條件也較成熟[88]。靈長(zhǎng)類動(dòng)物，與人同源性最高，并在情緒、社交等方面與人類最為類似，但存在研究周期長(zhǎng)、耗費(fèi)較高和動(dòng)物倫理等問題?？傊?，鼠模型經(jīng)濟(jì)成本、時(shí)間成本、技術(shù)條件、同源性和行為學(xué)觀察總體評(píng)估最優(yōu)?？筛鶕?jù)不同實(shí)驗(yàn)需要，選擇最優(yōu)的動(dòng)物模型。

6 展望

未來動(dòng)物模型研究的重點(diǎn)應(yīng)建立在其ASD基因型與表型關(guān)聯(lián)性的基礎(chǔ)上，從而能為更有效地研發(fā)和選擇治療方案打下基礎(chǔ)。目前ASD的干預(yù)療法多建立在改善行為的基礎(chǔ)上，未來的治療方向應(yīng)更傾向于分子療法。目前，針對(duì)Mecp2突變鼠，運(yùn)用基因修復(fù)技術(shù)能使其神經(jīng)損傷得到修復(fù)[88～90]，并且運(yùn)用病毒轉(zhuǎn)染Ube3a能使Angelman綜合征鼠模型的異常行為得到改善[91]。隨著越來越多ASD臨床資料的收集，并對(duì)ASD癥狀規(guī)范性的總結(jié)，輔之更深入地了解神經(jīng)系統(tǒng)的機(jī)制，再具備更理想的動(dòng)物模型，ASD將會(huì)被更加透徹的研究。

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(本文編輯：張崇凡)

10.3969/j.issn.1673-5501.2015.06.013

復(fù)旦大學(xué)附屬兒科醫(yī)院 上海，201102

徐秀，E-mail：xuxiu@shmu.edu.cn

2015-07-15

2015-11-20)