轉基因線蟲在阿爾茨海默癥研究中的應用

李　梵,羅劍鳴,劉少莉,韓　蕾,盧　江,張雅麗

(中國農業大學食品科學與營養工程學院,北京 100083)

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轉基因線蟲在阿爾茨海默癥研究中的應用

李梵,羅劍鳴,劉少莉,韓蕾,盧江,張雅麗*

(中國農業大學食品科學與營養工程學院,北京 100083)

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

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,在未來的十年這個數字還會增加[1]。大量的臨床實驗都在研究其病理學和病因學,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。研究人員設計了各種動物模型諸如β-淀粉樣蛋白模型等[2]、膽堿能系統損傷模型[3]、鋁模型[4]、tau蛋白模型[5]試圖尋找發病原因及機制,并取得了一定的研究成果。秀麗線蟲具有易培養、生長周期短、通體透明、體細胞數目一定、特定細胞位置恒定等優點。此外,秀麗線蟲基因組測序也在1998年完成。隨后,秀麗線蟲作為模式生物在細胞凋亡、神經性疾病等方面研究中應用更加廣泛。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中的研究應用。

1　阿爾茨海默病

AD是以進行性記憶減退、認知障礙與人格改變為主要臨床表現的一種神經系統變性疾病,也是老年人群中最主要的神經退行性疾病[6]。AD包括一系列神經損傷的癥狀,氧化損傷,炎癥和神經元機能障礙[7],并可見老年斑(Senile plaques,SP)與神經原纖維結(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的積累導致老年斑的形成。老年斑又稱淀粉樣斑(Amyloid Plaques,AP),主要由細胞外纖維和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刺激下小膠質細胞的活化、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、有害自由基的產生、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)及誘導型一氧化氮合酶(induced nitric oxide synthase,iNOS)等各種促炎因子的釋放,都直接或間接導致了神經系統的損傷[10]。大量體外實驗研究表明,Aβ聚集形成纖維或低聚物均比單體結構具有更強的神經毒性[11]。因此,尋找某種阻止Aβ聚集的抑制劑被認為是一種延緩AD的治療方案。

2　AD的秀麗線蟲模型

在研究線蟲的過程中,研究人員發現線蟲中含有人類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,因此需要通過轉基因的方式來得到AD模型。1995年,Link CD通過轉基因的方式建立了線蟲的Aβ模型[12]。轉基因線蟲模型用來研究AD毒性機理[13]、Aβ引起的基因表達的變化[14-17]及探究藥物抗AD的功效。

在眾多實驗室研究中,常用的轉基因線蟲品系主要為CL2006、CL4176等(表2)。其中CL4176使用肌肉細胞特異性啟動子myo-3來控制Aβ的表達,使線蟲表型上出現抽搐癥狀,復制了AD的癥狀和病理變化,從而可以明顯地區分轉基因的線蟲。CL4176線蟲不會在體內形成Aβ斑,因為Aβ在細胞內主要以水溶形式存在[18-19]。而CL2006線蟲使用啟動子unc-54來控制Aβ的表達,并引入表型基因rol-6。CL2006線蟲同CL4176線蟲一樣會出現肌肉細胞中Aβ沉積,此外還會有進行性麻痹的表現。

3　線蟲中AD的相關基因

3.1Aβ基因

雖然在轉基因線蟲中Aβ基因通過溫度誘導表達或是結構表達使線蟲體內Aβ大量形成,但Aβ基因的表達通常與β淀粉沉積并無直接聯系[20-22]。這可能是由于不同實驗所用轉基因線蟲品系不同,還與收集樣品時間相關。

3.2乙酰膽堿酯酶基因

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

3.3抗炎與應激相關基因

與神經性疾病相關的促進炎癥的基因及細胞素主要有TNFα,IL-1和IL-6。這些因子在不同的AD模型中已經證實與Aβ的神經毒性及由Aβ引起的AD發病機制有關[28-30]。Link CD等研究表明,在AD病人腦組織中TNFA1P1(TNFα-induced protein 1)表達上調。線蟲中有TNFA1P1的同源基因F22E5.6和ZC239.12。線蟲升溫培養后,CL4176體內F22E5.6和ZC239.12表達上調[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應激條件下促進其表達,增強抵抗力[31]。由于線蟲中熱激反應是由神經元主導的[32],HSP-16被認為是毒蛋白異常積累下的保護蛋白。在線蟲體內的HSP-16.2過表達會抑制β淀粉樣蛋白的毒性[33]。在AD病人腦組織中與應激相關的伴侶蛋白αB-crystallin(CRYAB)表達上調,而線蟲CL4176中CRYAB同源基因HSP16-2(γ46H3A.D)和HSP16-4(γ46H3A.E)在升溫培養后表達上調。六味地黃湯粗提物促進了熱激蛋白HSP-16.2的表達,使得線蟲癱瘓發生時間延后[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[35]。

3.4衰老相關基因

AD是與衰老相關的神經性疾病[36]。衰老相關基因主要有早衰老基因ps-1和ps-2,胰島素樣生長因子-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發病機理的復雜性,引起AD癥狀的上游神經遞質的缺失已經揭示[39],因此,越來越多的研究集中在開發新藥品上,以調節上游靶點,并由此揭示AD的發病機制。

4.1藥物研究

目前的治療及病情控制主要依賴于早發現,盡早服用一些有助于緩解與病情相關的藥物延遲病情的進一步惡化。

過去的十年中,許多藥物對AD有一定的緩解作用,特別是對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上主要的治療 AD 的藥物。美國 FDA批準的五種抗 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,提高患者的認知能力,但是并不能完全的治愈 AD,只能暫時改善癥狀,一定程度延緩病程,遠期療效不好,而且多有較為嚴重的毒副作用[41]。有些藥物會引起腸胃功能損害[28,42]。Memantine為一種美國食品藥品管理局批準用于治療中度和重度AD的藥物,可以通過削弱N-甲基-D-天冬氨酸感受器調節膽堿能代謝平衡,但也對腸胃有副作用[43]。

目前臨床上并沒有一種單靶點的藥物有效并安全。近年來,許多研究學者開始尋求多靶點藥物,同時對AD致病因子中幾個重要的作用靶點進行控制,這是一種高效的阻止AD神經退化進程的方法[44-48]。對此,有關體內研究實驗表明,memoquin[43]和IQM-622[49]作為多靶點是有效的。Donepezil和hupine Y[50]的合成物,對官能團進行修飾,與乙酰膽堿酶相互作用,起到類膽堿功能。另一方面,多靶點藥物結合乙酰膽堿酯酶使得乙酰膽堿酯酶無法再與Aβ結合[51-52],從而阻止了Aβ的聚合[53]。體外研究結果表明,合成物AVCRI104P4也能夠抑制丁酰膽堿酯酶,從而抑制Aβ的聚集[39]。多靶點研究藥物的設計考慮因素主要為多靶點藥物對多個靶點作用強度的問題,力求對各個靶標作用均衡[54]。目前,多靶點藥物仍在研究階段,諸多研究者期待通過化學合成的方法制備有效的多靶點藥物。

4.2植物提取物研究

近幾年來,國內外抗AD的研究熱點逐漸轉移到天然提取物上,一是由于AD病理復雜,并沒有某種單一藥物具有良好效果;二是因為其中一些藥物具有副作用。而在日常飲食中會攝入一些抗氧化劑,這些抗氧化劑對AD以及衰老相關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,大多是從中醫的角度出發篩選藥物并臨床實踐后發現的抗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。銀杏提取物對中樞神經系統的作用機理可能與氧化、清除脂質自由基,保護膜蛋白等作用有關[71]。用六味地黃湯的乙醇提取物(LWDH-EE)干預轉基因秀麗線蟲CL4176,發現其能有效延緩Aβ1-42誘導的秀麗線蟲麻痹癥狀[34]。在胡桃酮誘導CL4176 氧化應激后,LWDH-EE能降低其體內活性氧的產生,在熱應激后,LWDH-EE能減少CL4176中amy-1基因表達,同時升高HSP的表達。這說明LWDH-EE通過抗氧化、調節熱休克蛋白的途徑來緩解β淀粉樣蛋白產生的毒性作用[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雜性,很難解釋清楚抗AD的作用機制和有效成分。同時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行抗AD發病機制的高水平研究。

單體物質也可以從結構上的相似性推斷功能的相似性,丹酚酸與姜黃素結構上相似,姜黃素具有抗Aβ功能,通過實驗證明丹酚酸也有抗Aβ聚集功能[72]。分子動力學模擬表明,丹酚酸與Aβ的C端結合從而穩定了Aβ的α螺旋結構。小分子可以與Aβ的C端結合使得構象由α螺旋變為β折疊[73-74]。體外實驗研究顯示,單體的取代基和空間構象共同影響抗Aβ活性[75]。但體外實驗與體內實驗并不完全一致,LWDH體外實驗沒有抑制Aβ聚集,在體內實驗中卻是有效的[34]。不同的單體作用機理不同。雖然部分生物堿也有抗氧化性質,但在線蟲實驗中,抗氧化性質在抗AD的過程并不是完全必需的。Rong Di等研究結果表明Haemanthidine等四種生物堿具有抑制Aβ誘導毒性的作用,而且通過ROS驗證后發現,這4種生物堿對緩解Aβ誘導毒性的原因與抗氧化作用無關,可能是其它直接作用于Aβ及其相關基因上的途徑來起到緩解Aβ誘導毒性的作用[27]。通過線蟲癱瘓實驗可以看出,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。然而,Kumar等指出水飛薊素抗AD與抗氧化有關[76]。表2總結了以秀麗線蟲為研究工具能夠對AD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的作用機理,但植物粗提物中多種物質的存在會相互影響,共同作用于AD致病因子,從而降低致病因子對機體的損害。綠茶提取物中水溶性VC與脂溶性VE成分之間會相互促進,更好地發揮抗AD的功能,而VE單獨作用時效果不明顯[68]。

5　展望

模式動物為解決天然產物對神經性疾病的治療提供了便利。轉基因線蟲作為模式動物,用來探究Aβ的毒性的分子機理,在植物提取物抗氧化物乃至藥物的篩選方面發揮著重要的作用,線蟲已經成為研究衰老及與衰老相關的神經性疾病的良好模型。利用線蟲已闡明了植物提取物在體內的抗AD部分機制,能夠很好地促進該研究領域向高等動物繼續深化,直至發展成為臨床上治療氧化應激相關疾病首選的純天然藥物。

雖然線蟲模型已經取得了一定成果,但是線蟲作為一種低級動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物中實驗。其次,AD病因復雜,發病機制并未研究透徹,目前發現的與AD有關的基因位點很有限,發現新位點,明確更多與AD相關的基因才能更好地研發治療藥物。

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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。

現代農業產業技術體系專項資金資助(CARS-30-yz-2)。

TS201.1

A

1002-0306(2016)07-0361-07

10.13386/j.issn1002-0306.2016.07.062