轉(zhuǎn)基因線蟲在阿爾茨海默癥研究中的應(yīng)用

李　梵,羅劍鳴,劉少莉,韓　蕾,盧　江,張雅麗

(中國(guó)農(nóng)業(yè)大學(xué)食品科學(xué)與營(yíng)養(yǎng)工程學(xué)院,北京 100083)

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轉(zhuǎn)基因線蟲在阿爾茨海默癥研究中的應(yīng)用

李梵,羅劍鳴,劉少莉,韓蕾,盧江,張雅麗*

(中國(guó)農(nóng)業(yè)大學(xué)食品科學(xué)與營(yíng)養(yǎng)工程學(xué)院,北京 100083)

由于秀麗線蟲基因與人類基因的同源性高達(dá)40%,其神經(jīng)元的功能和神經(jīng)遞質(zhì)與人類的也高度類似,因而秀麗線蟲成為研究神經(jīng)性疾病機(jī)制的有效模型。本文就轉(zhuǎn)基因秀麗線蟲在阿爾茨海默病中的研究進(jìn)展進(jìn)行了闡述,總結(jié)了近年來天然提取物在阿爾茨海默癥中的應(yīng)用,并對(duì)以轉(zhuǎn)基因線蟲為模型進(jìn)行研究的單體進(jìn)行了闡述。

秀麗線蟲,阿爾茨海默癥,天然提取物,單體

阿爾茨海默病(Alzheimer’sdisease,AD)是世界上引起死亡最多的十種疾病之一,世界上四千萬人患病,在未來的十年這個(gè)數(shù)字還會(huì)增加[1]。大量的臨床實(shí)驗(yàn)都在研究其病理學(xué)和病因?qū)W,除了一些緩解癥狀的藥在使用之外,AD的病因及治愈并沒有被完全闡述。研究人員設(shè)計(jì)了各種動(dòng)物模型諸如β-淀粉樣蛋白模型等[2]、膽堿能系統(tǒng)損傷模型[3]、鋁模型[4]、tau蛋白模型[5]試圖尋找發(fā)病原因及機(jī)制,并取得了一定的研究成果。秀麗線蟲具有易培養(yǎng)、生長(zhǎng)周期短、通體透明、體細(xì)胞數(shù)目一定、特定細(xì)胞位置恒定等優(yōu)點(diǎn)。此外,秀麗線蟲基因組測(cè)序也在1998年完成。隨后,秀麗線蟲作為模式生物在細(xì)胞凋亡、神經(jīng)性疾病等方面研究中應(yīng)用更加廣泛。其中,在秀麗線蟲中建立的AD模型主要有Aβ模型和tau模型兩種。本文主要介紹Aβ模型線蟲在AD中的研究應(yīng)用。

1　阿爾茨海默病

AD是以進(jìn)行性記憶減退、認(rèn)知障礙與人格改變?yōu)橹饕R床表現(xiàn)的一種神經(jīng)系統(tǒng)變性疾病,也是老年人群中最主要的神經(jīng)退行性疾病[6]。AD包括一系列神經(jīng)損傷的癥狀,氧化損傷,炎癥和神經(jīng)元機(jī)能障礙[7],并可見老年斑(Senile plaques,SP)與神經(jīng)原纖維結(jié)(Neurofibrillary tangles,NFT)等病變[8]。AD的早期病理是大腦中β樣淀粉蛋白前體代謝成為Aβ蛋白[9],Aβ的積累導(dǎo)致老年斑的形成。老年斑又稱淀粉樣斑(Amyloid Plaques,AP),主要由細(xì)胞外纖維和β淀粉樣蛋白(beta-amyloid,Aβ)形成沉淀引起。目前Aβ是比較明確的致病因子。Aβ刺激下小膠質(zhì)細(xì)胞的活化、還原型煙酰胺腺嘌呤二核苷酸(NADPH)氧化酶的激活、大量活性氧的生成、有害自由基的產(chǎn)生、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)及誘導(dǎo)型一氧化氮合酶(induced nitric oxide synthase,iNOS)等各種促炎因子的釋放,都直接或間接導(dǎo)致了神經(jīng)系統(tǒng)的損傷[10]。大量體外實(shí)驗(yàn)研究表明,Aβ聚集形成纖維或低聚物均比單體結(jié)構(gòu)具有更強(qiáng)的神經(jīng)毒性[11]。因此,尋找某種阻止Aβ聚集的抑制劑被認(rèn)為是一種延緩AD的治療方案。

2　AD的秀麗線蟲模型

在研究線蟲的過程中,研究人員發(fā)現(xiàn)線蟲中含有人類APP類似基因apl-1,它在線蟲中是跨膜蛋白,但是它不含Aβ序列,因此需要通過轉(zhuǎn)基因的方式來得到AD模型。1995年,Link CD通過轉(zhuǎn)基因的方式建立了線蟲的Aβ模型[12]。轉(zhuǎn)基因線蟲模型用來研究AD毒性機(jī)理[13]、Aβ引起的基因表達(dá)的變化[14-17]及探究藥物抗AD的功效。

在眾多實(shí)驗(yàn)室研究中,常用的轉(zhuǎn)基因線蟲品系主要為CL2006、CL4176等(表2)。其中CL4176使用肌肉細(xì)胞特異性啟動(dòng)子myo-3來控制Aβ的表達(dá),使線蟲表型上出現(xiàn)抽搐癥狀,復(fù)制了AD的癥狀和病理變化,從而可以明顯地區(qū)分轉(zhuǎn)基因的線蟲。CL4176線蟲不會(huì)在體內(nèi)形成Aβ斑,因?yàn)锳β在細(xì)胞內(nèi)主要以水溶形式存在[18-19]。而CL2006線蟲使用啟動(dòng)子unc-54來控制Aβ的表達(dá),并引入表型基因rol-6。CL2006線蟲同CL4176線蟲一樣會(huì)出現(xiàn)肌肉細(xì)胞中Aβ沉積,此外還會(huì)有進(jìn)行性麻痹的表現(xiàn)。

3　線蟲中AD的相關(guān)基因

3.1Aβ基因

雖然在轉(zhuǎn)基因線蟲中Aβ基因通過溫度誘導(dǎo)表達(dá)或是結(jié)構(gòu)表達(dá)使線蟲體內(nèi)Aβ大量形成,但Aβ基因的表達(dá)通常與β淀粉沉積并無直接聯(lián)系[20-22]。這可能是由于不同實(shí)驗(yàn)所用轉(zhuǎn)基因線蟲品系不同,還與收集樣品時(shí)間相關(guān)。

3.2乙酰膽堿酯酶基因

膽堿能缺失學(xué)說認(rèn)為AD發(fā)病是源于病人體內(nèi)乙酰膽堿的缺失[23]。由于線蟲基因的保守性,可以通過檢測(cè)線蟲乙酰膽堿酯酶的表達(dá)量觀察給藥的效果。與脊椎動(dòng)物只有一個(gè)乙酰膽堿酯酶基因不同,線蟲有4種乙酰膽堿酶基因,ace-1在所有的體細(xì)胞和外陰肌肉細(xì)胞中表達(dá)[24],ace-2僅在神經(jīng)細(xì)胞中表達(dá)[25],ace-3則是在眼部肌肉細(xì)胞和神經(jīng)細(xì)胞中表達(dá),ace-4基本檢測(cè)不到[26]。但ACE-1、ACE-2占了酶活的95%,ACE-3占了5%,ACE-4基本檢測(cè)不到酶活[27]。

3.3抗炎與應(yīng)激相關(guān)基因

與神經(jīng)性疾病相關(guān)的促進(jìn)炎癥的基因及細(xì)胞素主要有TNFα,IL-1和IL-6。這些因子在不同的AD模型中已經(jīng)證實(shí)與Aβ的神經(jīng)毒性及由Aβ引起的AD發(fā)病機(jī)制有關(guān)[28-30]。Link CD等研究表明,在AD病人腦組織中TNFA1P1(TNFα-induced protein 1)表達(dá)上調(diào)。線蟲中有TNFA1P1的同源基因F22E5.6和ZC239.12。線蟲升溫培養(yǎng)后,CL4176體內(nèi)F22E5.6和ZC239.12表達(dá)上調(diào)[30]。

熱激蛋白(heat shock proteins,HSPs)是一類類似分子伴侶的小分子多肽,在Aβ應(yīng)激條件下促進(jìn)其表達(dá),增強(qiáng)抵抗力[31]。由于線蟲中熱激反應(yīng)是由神經(jīng)元主導(dǎo)的[32],HSP-16被認(rèn)為是毒蛋白異常積累下的保護(hù)蛋白。在線蟲體內(nèi)的HSP-16.2過表達(dá)會(huì)抑制β淀粉樣蛋白的毒性[33]。在AD病人腦組織中與應(yīng)激相關(guān)的伴侶蛋白αB-crystallin(CRYAB)表達(dá)上調(diào),而線蟲CL4176中CRYAB同源基因HSP16-2(γ46H3A.D)和HSP16-4(γ46H3A.E)在升溫培養(yǎng)后表達(dá)上調(diào)。六味地黃湯粗提物促進(jìn)了熱激蛋白HSP-16.2的表達(dá),使得線蟲癱瘓發(fā)生時(shí)間延后[34]。有研究表明姜黃素能夠抑制IL-1、TNFα、COX-2、iNOS和NF-κB等炎癥因子的表達(dá)[35]。

3.4衰老相關(guān)基因

AD是與衰老相關(guān)的神經(jīng)性疾病[36]。衰老相關(guān)基因主要有早衰老基因ps-1和ps-2,胰島素樣生長(zhǎng)因子-1基因等基因。在線蟲中,與ps-1和ps-2同源的基因是sel-12[37]和hop-1[38]。

4　AD的治療

由于AD發(fā)病機(jī)理的復(fù)雜性,引起AD癥狀的上游神經(jīng)遞質(zhì)的缺失已經(jīng)揭示[39],因此,越來越多的研究集中在開發(fā)新藥品上,以調(diào)節(jié)上游靶點(diǎn),并由此揭示AD的發(fā)病機(jī)制。

4.1藥物研究

目前的治療及病情控制主要依賴于早發(fā)現(xiàn),盡早服用一些有助于緩解與病情相關(guān)的藥物延遲病情的進(jìn)一步惡化。

過去的十年中,許多藥物對(duì)AD有一定的緩解作用,特別是對(duì)Aβ的形成或是Aβ的沉積[40]。乙酰膽堿酯酶抑制劑是目前市場(chǎng)上主要的治療 AD 的藥物。美國(guó) FDA批準(zhǔn)的五種抗 AD 藥物中乙酰膽堿酯酶抑制劑占了四種,它們是tacrine,donepezil,rivastigmine,galanthamine,這些藥物能夠改善患者的癥狀,提高患者的認(rèn)知能力,但是并不能完全的治愈 AD,只能暫時(shí)改善癥狀,一定程度延緩病程,遠(yuǎn)期療效不好,而且多有較為嚴(yán)重的毒副作用[41]。有些藥物會(huì)引起腸胃功能損害[28,42]。Memantine為一種美國(guó)食品藥品管理局批準(zhǔn)用于治療中度和重度AD的藥物,可以通過削弱N-甲基-D-天冬氨酸感受器調(diào)節(jié)膽堿能代謝平衡,但也對(duì)腸胃有副作用[43]。

目前臨床上并沒有一種單靶點(diǎn)的藥物有效并安全。近年來,許多研究學(xué)者開始尋求多靶點(diǎn)藥物,同時(shí)對(duì)AD致病因子中幾個(gè)重要的作用靶點(diǎn)進(jìn)行控制,這是一種高效的阻止AD神經(jīng)退化進(jìn)程的方法[44-48]。對(duì)此,有關(guān)體內(nèi)研究實(shí)驗(yàn)表明,memoquin[43]和IQM-622[49]作為多靶點(diǎn)是有效的。Donepezil和hupine Y[50]的合成物,對(duì)官能團(tuán)進(jìn)行修飾,與乙酰膽堿酶相互作用,起到類膽堿功能。另一方面,多靶點(diǎn)藥物結(jié)合乙酰膽堿酯酶使得乙酰膽堿酯酶無法再與Aβ結(jié)合[51-52],從而阻止了Aβ的聚合[53]。體外研究結(jié)果表明,合成物AVCRI104P4也能夠抑制丁酰膽堿酯酶,從而抑制Aβ的聚集[39]。多靶點(diǎn)研究藥物的設(shè)計(jì)考慮因素主要為多靶點(diǎn)藥物對(duì)多個(gè)靶點(diǎn)作用強(qiáng)度的問題,力求對(duì)各個(gè)靶標(biāo)作用均衡[54]。目前,多靶點(diǎn)藥物仍在研究階段,諸多研究者期待通過化學(xué)合成的方法制備有效的多靶點(diǎn)藥物。

4.2植物提取物研究

近幾年來,國(guó)內(nèi)外抗AD的研究熱點(diǎn)逐漸轉(zhuǎn)移到天然提取物上,一是由于AD病理復(fù)雜,并沒有某種單一藥物具有良好效果;二是因?yàn)槠渲幸恍┧幬锞哂懈弊饔?。而在日常飲食中?huì)攝入一些抗氧化劑,這些抗氧化劑對(duì)AD以及衰老相關(guān)的疾病具有一定的影響及作用。表1顯示的為幾種植物提取物的研究成果。

從表1中可以看出,很多為中藥成分,大多是從中醫(yī)的角度出發(fā)篩選藥物并臨床實(shí)踐后發(fā)現(xiàn)的抗AD候選藥物。其中,銀杏提取物EGb761開啟了植物提取物研究的熱潮。銀杏提取物對(duì)中樞神經(jīng)系統(tǒng)的作用機(jī)理可能與氧化、清除脂質(zhì)自由基,保護(hù)膜蛋白等作用有關(guān)[71]。用六味地黃湯的乙醇提取物(LWDH-EE)干預(yù)轉(zhuǎn)基因秀麗線蟲CL4176,發(fā)現(xiàn)其能有效延緩Aβ1-42誘導(dǎo)的秀麗線蟲麻痹癥狀[34]。在胡桃酮誘導(dǎo)CL4176 氧化應(yīng)激后,LWDH-EE能降低其體內(nèi)活性氧的產(chǎn)生,在熱應(yīng)激后,LWDH-EE能減少CL4176中amy-1基因表達(dá),同時(shí)升高HSP的表達(dá)。這說明LWDH-EE通過抗氧化、調(diào)節(jié)熱休克蛋白的途徑來緩解β淀粉樣蛋白產(chǎn)生的毒性作用[34]。

表1　植物提取物抗AD及衰老研究

4.3單體研究

由于植物粗提物成分的復(fù)雜性,很難解釋清楚抗AD的作用機(jī)制和有效成分。同時(shí)由于單體提取制備的困難性,目前只有一部分單體被用來進(jìn)行抗AD發(fā)病機(jī)制的高水平研究。

單體物質(zhì)也可以從結(jié)構(gòu)上的相似性推斷功能的相似性,丹酚酸與姜黃素結(jié)構(gòu)上相似,姜黃素具有抗Aβ功能,通過實(shí)驗(yàn)證明丹酚酸也有抗Aβ聚集功能[72]。分子動(dòng)力學(xué)模擬表明,丹酚酸與Aβ的C端結(jié)合從而穩(wěn)定了Aβ的α螺旋結(jié)構(gòu)。小分子可以與Aβ的C端結(jié)合使得構(gòu)象由α螺旋變?yōu)棣抡郫B[73-74]。體外實(shí)驗(yàn)研究顯示,單體的取代基和空間構(gòu)象共同影響抗Aβ活性[75]。但體外實(shí)驗(yàn)與體內(nèi)實(shí)驗(yàn)并不完全一致,LWDH體外實(shí)驗(yàn)沒有抑制Aβ聚集,在體內(nèi)實(shí)驗(yàn)中卻是有效的[34]。不同的單體作用機(jī)理不同。雖然部分生物堿也有抗氧化性質(zhì),但在線蟲實(shí)驗(yàn)中,抗氧化性質(zhì)在抗AD的過程并不是完全必需的。Rong Di等研究結(jié)果表明Haemanthidine等四種生物堿具有抑制Aβ誘導(dǎo)毒性的作用,而且通過ROS驗(yàn)證后發(fā)現(xiàn),這4種生物堿對(duì)緩解Aβ誘導(dǎo)毒性的原因與抗氧化作用無關(guān),可能是其它直接作用于Aβ及其相關(guān)基因上的途徑來起到緩解Aβ誘導(dǎo)毒性的作用[27]。通過線蟲癱瘓實(shí)驗(yàn)可以看出,這4種生物堿與治療老年癡呆癥藥物memantine相比,四種生物堿的抑制作用更強(qiáng)。然而,Kumar等指出水飛薊素抗AD與抗氧化有關(guān)[76]。表2總結(jié)了以秀麗線蟲為研究工具能夠?qū)D有緩解作用的單體研究。

表2　目前所研究抗AD的單體

雖然單體研究有助于確定某一物質(zhì)的作用機(jī)理,但植物粗提物中多種物質(zhì)的存在會(huì)相互影響,共同作用于AD致病因子,從而降低致病因子對(duì)機(jī)體的損害。綠茶提取物中水溶性VC與脂溶性VE成分之間會(huì)相互促進(jìn),更好地發(fā)揮抗AD的功能,而VE單獨(dú)作用時(shí)效果不明顯[68]。

5　展望

模式動(dòng)物為解決天然產(chǎn)物對(duì)神經(jīng)性疾病的治療提供了便利。轉(zhuǎn)基因線蟲作為模式動(dòng)物,用來探究Aβ的毒性的分子機(jī)理,在植物提取物抗氧化物乃至藥物的篩選方面發(fā)揮著重要的作用,線蟲已經(jīng)成為研究衰老及與衰老相關(guān)的神經(jīng)性疾病的良好模型。利用線蟲已闡明了植物提取物在體內(nèi)的抗AD部分機(jī)制,能夠很好地促進(jìn)該研究領(lǐng)域向高等動(dòng)物繼續(xù)深化,直至發(fā)展成為臨床上治療氧化應(yīng)激相關(guān)疾病首選的純天然藥物。

雖然線蟲模型已經(jīng)取得了一定成果,但是線蟲作為一種低級(jí)動(dòng)物只能作為天然藥物的初期篩選,之后仍需要在老鼠或猴子等高等動(dòng)物中實(shí)驗(yàn)。其次,AD病因復(fù)雜,發(fā)病機(jī)制并未研究透徹,目前發(fā)現(xiàn)的與AD有關(guān)的基因位點(diǎn)很有限,發(fā)現(xiàn)新位點(diǎn),明確更多與AD相關(guān)的基因才能更好地研發(fā)治療藥物。

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Application of transgenic Caenorhabditis elegans in Alzheimer’s disease

LI Fan,LUO Jian-ming,LIU Shao-li,HAN Lei,LU Jiang,ZHANG Ya-li*

(College of Food Science and Nutritional Engineering,China Agriculture University,Beijing 100083,China)

Since the Caenorhabditis elegans genome displayes high homology(40%)to human genome and its functions of neurons and neurotransmitters are also highly similar with those of humans,Caenorhabditis elegans have become an effective model of the neural mechanisms of disease.Researches of applications of transgenic Caenorhabditis elegans in Alzheimer’s disease were focused on and the applications of natural extracts in Alzheimer’s disease in recent years were summarized.Also some monomers that were applied in transgenic Caenorhabditis elegans were summed up.

Caenorhabditis elegans;Alzheimer’s disease;naturalextracts;monomer

2015-08-27

李梵(1992-),女,碩士,研究方向:葡萄與葡萄酒工程，E-mail:lifan1992@cau.edu.cn。

張雅麗(1975-),女,博士,副教授,研究方向:葡萄與葡萄酒工程，E-mail:zhangyali@cau.edu.cn。

現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系專項(xiàng)資金資助(CARS-30-yz-2)。

TS201.1

A

1002-0306(2016)07-0361-07

10.13386/j.issn1002-0306.2016.07.062