第一臨床醫(yī)學(xué)院郭軍紅課題組聯(lián)合第三軍醫(yī)大學(xué)王延江教授課題組提出阿爾茨海默病體液生物標(biāo)志物的新觀點(diǎn)
2022年3月19日，來(lái)自山西醫(yī)科大學(xué)第一臨床醫(yī)學(xué)院的郭軍紅教授及黃珊博士研究生聯(lián)合第三軍醫(yī)大學(xué)的王延江教授在Neuroscience Bulletin（IF=5.203）上發(fā)表了題為“Biofluid biomarkers of Alzheimer’s disease: progress, problems, and perspectives.”的最新文章。該文章圍繞AD外周體液生物標(biāo)志物，首先介紹了A-T-N-X框架的建立過(guò)程及過(guò)去普遍存在的問(wèn)題。針對(duì)A-T-N-X框架的具體內(nèi)容進(jìn)行了深入解讀，剖析其中存在的優(yōu)勢(shì)及挑戰(zhàn)，并系統(tǒng)總結(jié)了相應(yīng)的解決方案，為AD生物標(biāo)志物的進(jìn)一步研究探索提供了方向。最后，基于目前的生物標(biāo)志物檢測(cè)手段及研究現(xiàn)狀，作者提出了更為全面的AD診斷體系，同時(shí)也為今后AD及其他神經(jīng)退行性疾病生物標(biāo)志物的研究與應(yīng)用開(kāi)辟了新的方向。
阿爾茨海默?。?/span>Alzheimer’s disease, AD）是一種進(jìn)行性神經(jīng)退行性疾病，是最常見(jiàn)的癡呆癥類型，以β-淀粉樣蛋白（Aβ）斑塊形成、細(xì)胞內(nèi)Tau蛋白聚集、神經(jīng)元和突觸丟失等病理表現(xiàn)為特征。隨著人口老齡化的進(jìn)展，AD發(fā)病率逐年增加，給社會(huì)帶來(lái)巨大的經(jīng)濟(jì)負(fù)擔(dān)。目前，對(duì)AD的診斷仍存在困難，生物標(biāo)志物對(duì)于準(zhǔn)確和早期識(shí)別AD至關(guān)重要，也是有效治療該病的先決條件。
AD生物標(biāo)志物A/T/N框架在2016年由Clifford等人首次提出，2018年被國(guó)際老齡化和阿爾茨海默病協(xié)會(huì)接受并推廣。“A”是指Aβ生物標(biāo)志物（淀粉樣蛋白PET或腦脊液Aβ-42），“T”是Tau生物標(biāo)志物（腦脊液磷酸化Tau（p-tau）或p-Tau PET），“N”是神經(jīng)變性或神經(jīng)元損傷的生物標(biāo)志物（[18F]-氟脫氧葡萄糖 PET、結(jié)構(gòu)磁共振或腦脊液總Tau（t-tau））。這一臨床生物學(xué)框架描繪了AD的病理生理特征，增加了AD診斷的準(zhǔn)確性。然而，現(xiàn)有的框架很難對(duì)AD的病理改變提供全面的解釋，許多重要的神經(jīng)損傷標(biāo)志物未被包含其中。同時(shí)，腦脊液檢查的有創(chuàng)性及PET掃描價(jià)格昂貴且輻射暴露的特點(diǎn)限制了該診斷框架的應(yīng)用。因此，AD外周生物標(biāo)志物框架的完善至關(guān)重要。
外周體液A-T-N-X框架的建立
由于腦脊液檢查具有有創(chuàng)性，同時(shí)PET掃描技術(shù)價(jià)格昂貴且具有輻射暴露，因此，外周生物標(biāo)志物的研究對(duì)AD診斷具有重要價(jià)值。目前研究人員發(fā)現(xiàn)一些血漿生物標(biāo)志物，在診斷AD的特異性和靈敏性與腦脊液檢查和PET掃描相當(dāng)。然而，血漿生物標(biāo)志物的推廣存在巨大的困難。①只有來(lái)自中樞神經(jīng)系統(tǒng)（CNS）的一小部分生物標(biāo)志物能夠通過(guò)血腦屏障（BBB）、蛛網(wǎng)膜顆粒、類淋巴系統(tǒng)和血管系統(tǒng)進(jìn)入外周體液系統(tǒng)，進(jìn)而在血液中被稀釋，并且在復(fù)雜的血液背景中，生物標(biāo)志物可以被酶降解，或與各種血液蛋白或血細(xì)胞形成復(fù)合體，這些因素阻礙了生物標(biāo)志物的準(zhǔn)確檢測(cè)；②肝臟和腎臟以及相關(guān)器官的巨噬細(xì)胞可以清除生物標(biāo)志物，一些外周組織可能會(huì)產(chǎn)生相同的生物標(biāo)志物并釋放到血液中；③由于新陳代謝、飲食和藥物等因素的不同，外周生物標(biāo)志物的水平在個(gè)體之間波動(dòng)，同時(shí)生物標(biāo)志物水平隨不同疾病的不同時(shí)期也會(huì)存在波動(dòng)。所有這些因素都阻礙了血漿生物標(biāo)志物與大腦中對(duì)應(yīng)的生物標(biāo)志物之間的相關(guān)性。針對(duì)這些存在的問(wèn)題有一些解決方案。首先，開(kāi)發(fā)超敏感技術(shù)可以擴(kuò)大血漿生物標(biāo)志物的檢測(cè)范圍。同時(shí)，新開(kāi)發(fā)的抗體在捕獲生物標(biāo)志物方面更具特異性和敏感性。在血液中濃縮生物標(biāo)志物的樣本處理方法也可以解決稀釋效應(yīng)。其次，血液神經(jīng)元源性外泌體（NDEs）是從CNS特異性衍生出來(lái)的，故NDEs可較為特異性反應(yīng)CNS中生物標(biāo)志物水平，同時(shí)，NDEs中的生物標(biāo)志物可以減少血液中的干擾，保護(hù)內(nèi)容物不被降解。第三，不同的采血位置可能會(huì)影響檢測(cè)結(jié)果。例如，頸內(nèi)靜脈可能是削弱外周器官清除和血液稀釋效果的最佳采血點(diǎn)。第四，BBB紊亂在AD中很常見(jiàn)，其嚴(yán)重程度因疾病分期和個(gè)體因素而異。用統(tǒng)一的方法評(píng)價(jià)BBB通透性有助于更準(zhǔn)確地分析外周A-T-N-X系統(tǒng)。除了BBB外，生物標(biāo)志物從CNS到外周體液的途徑仍不完全清楚，需要進(jìn)一步研究探討。最后，對(duì)AD患者的體液采集方法、采集時(shí)間和血漿生物標(biāo)志物檢測(cè)方法進(jìn)行統(tǒng)一能夠有效解決個(gè)體及時(shí)相差異性的問(wèn)題。
除血漿生物標(biāo)志物外，許多其他外周生物標(biāo)志物也在積極研究中，為AD的診斷及篩查提供更多可能性。
A-T-N-X框架的內(nèi)容
Aβ
Aβ是一種由36~43個(gè)氨基酸組成的多肽，由淀粉樣前體蛋白(APP)通過(guò)β-分泌酶和γ-分泌酶剪切而來(lái)。Aβ是AD的中樞生物標(biāo)志物，同時(shí)也是淀粉樣斑塊的主要成分。腦脊液中Aβ40、Aβ42及其比值和PET檢測(cè)淀粉樣蛋白已成為AD診斷的重要手段。為了解決其有創(chuàng)性及高成本的缺陷，研究人員提出血漿Aβ檢測(cè)。然而，血漿Aβ作為AD生物標(biāo)志物仍存在困難。首先，Aβ粘性較高，很難向外周轉(zhuǎn)運(yùn)。其次，由于血液的稀釋等因素，目前的檢測(cè)手段很難準(zhǔn)確測(cè)定血液中的可溶性Aβ水平。同時(shí)，外周也會(huì)產(chǎn)生Aβ，且血液中多種蛋白及細(xì)胞與Aβ結(jié)合可掩蓋其抗原表位使其不易被抗體捕獲，降低血液Aβ診斷AD的特異性。此外，前述測(cè)定NDEs中的Aβ只能反映CNS中細(xì)胞內(nèi)Aβ水平，而AD病理主要體現(xiàn)在細(xì)胞外淀粉樣斑塊的形成，因此，NDEs中的Aβ無(wú)法直接反映AD病理特征。這些問(wèn)題的存在給外周Aβ作為AD生物標(biāo)志物帶來(lái)巨大的挑戰(zhàn)。為此，首先要探究Aβ外周轉(zhuǎn)運(yùn)的機(jī)制及其影響因素。其次，檢測(cè)前的預(yù)處理能夠降低血液中各種因素的干擾，如ELISA檢測(cè)Aβ水平前，對(duì)血漿樣本進(jìn)行蛋白變性處理，能夠使與蛋白結(jié)合的Aβ釋放出來(lái)，提高結(jié)果的準(zhǔn)確性。同時(shí)，AD患者的基因表達(dá)在中樞和外周存在差異，有助于區(qū)分Aβ的來(lái)源，從而幫助準(zhǔn)確測(cè)定與AD病理相關(guān)的外周Aβ水平。
Tau
Tau蛋白是微管相關(guān)蛋白Tau（MAPT）基因的產(chǎn)物，具有穩(wěn)定微管的生理功能。病理性Tau被認(rèn)為是Aβ的下游蛋白，反映神經(jīng)元的損傷程度。病理性Tau存在多種翻譯后修飾（Post-translational modifications, PTMs），包括磷酸化、乙酰化、甲基化、泛素化、糖基化和硝化等。同時(shí)，PTM存在不同的修飾位點(diǎn)，特殊的PTM位點(diǎn)與AD病理相關(guān)，有助于AD的診斷。PTM中最常見(jiàn)的是磷酸化Tau，p-Tau是神經(jīng)纖維纏結(jié)的主要成分。其中，腦脊液及血液中的p-tau217、p-tau231和p-tau181對(duì)AD的特異性較高。P-tau217、P-tau181等在AD無(wú)癥狀階段特異性升高，并隨著AD的進(jìn)展而變化，從而有助于AD早期診斷、鑒別診斷、病情變化監(jiān)測(cè)及預(yù)后判斷。然而，在不同的研究中，p-tau水平對(duì)AD診斷的特異性存在差異，這可能與檢測(cè)前的預(yù)處理、檢測(cè)方法及使用的試劑種類有關(guān)。
神經(jīng)退行性變的生物標(biāo)志物
神經(jīng)絲輕鏈（neurofilament light chain, NFL）作為軸突骨架的組成部分，是反映軸突變性的生物標(biāo)志物，在臨床癥狀出現(xiàn)前就已出現(xiàn)其水平的改變，同時(shí)隨著AD的進(jìn)展發(fā)生變化；T-tau是神經(jīng)變性的生物標(biāo)志物，反映神經(jīng)元分泌Tau和皮質(zhì)厚度的非特異性變化；類視錐蛋白-1（visinin-like protein 1, VILIP-1）是一種在神經(jīng)元中表達(dá)的鈣敏感蛋白，反映神經(jīng)元損傷。然而，這些生物標(biāo)志物缺乏特異性，在應(yīng)用時(shí)需要與AD特異性生物標(biāo)志物，如Aβ等聯(lián)合應(yīng)用以提高診斷的準(zhǔn)確性。
“X”生物標(biāo)志物
“X”指神經(jīng)免疫失調(diào)、突觸功能障礙和BBB改變等其他AD可能發(fā)病機(jī)制中涉及的生物標(biāo)志物，在A/T/N框架中加入“X”能夠更加全面反映AD病理變化，闡明AD的發(fā)病機(jī)制。我們將“X”分為兩部分，分別是中樞X（XC），即與突觸損傷、神經(jīng)膠質(zhì)細(xì)胞、神經(jīng)炎癥和免疫等相關(guān)的生物標(biāo)記物，和外周X（XP），即與系統(tǒng)免疫、炎癥和新陳代謝等相關(guān)的生物標(biāo)記物。
突觸功能障礙的生物標(biāo)志物
突觸是學(xué)習(xí)和記憶的基本結(jié)構(gòu)，突觸丟失與認(rèn)知能力下降有關(guān)。反映突觸功能障礙的生物標(biāo)志物有樹(shù)突狀蛋白神經(jīng)顆粒素（dendritic protein neurogranin, Ng）、突觸前蛋白，如神經(jīng)調(diào)節(jié)素（neuromodulin, GAP43）、突觸體相關(guān)蛋白25（Synaptosomalassociated protein 25 , SNAP25）及突觸結(jié)合蛋白等。
神經(jīng)膠質(zhì)細(xì)胞、神經(jīng)免疫和神經(jīng)炎癥的生物標(biāo)志物
AD的發(fā)病與星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞的激活密切相關(guān)，星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞的生物標(biāo)志物與AD有關(guān)。其中具有代表性的有膠質(zhì)纖維酸性蛋白（glial fibrillary acidic protein, GFAP）、S100B、幾丁質(zhì)酶-3樣蛋白1（chitinase-3-like protein 1, YKL-40）、髓樣細(xì)胞觸發(fā)受體2（triggering receptor expressed on myeloid cells-2 , TREM2）及MicroRNA-425等。
系統(tǒng)免疫、炎癥和代謝的生物標(biāo)志物
一些非特異性外周生物標(biāo)志物，如腫瘤壞死因子、白細(xì)胞介素2、免疫球蛋白和補(bǔ)體家族，可用于評(píng)估AD的炎癥狀態(tài)。AD常伴隨有多種代謝紊亂性疾病，相應(yīng)的血漿代謝物包括葡萄糖、血脂、氨基酸、維生素和微量元素，都與AD有關(guān)。高水平的膽固醇和甘油三酯與AD相關(guān)。血液中同型半胱氨酸水平較高，維生素A、B12、C、D、E和葉酸水平較低，與MCI和AD相關(guān)。這些生物標(biāo)志物對(duì)診斷AD的特異性不高，但能夠與AD特異性生物標(biāo)志物如Aβ等協(xié)同應(yīng)用提高診斷的準(zhǔn)確性及檢出率。
AD生物標(biāo)志物的檢測(cè)技術(shù)
在AD中最廣泛使用的生物標(biāo)志物分析技術(shù)是質(zhì)譜（MS）分析和免疫檢測(cè)。近來(lái)，越來(lái)越多的檢測(cè)技術(shù)出現(xiàn)，提高了生物標(biāo)志物的檢測(cè)準(zhǔn)確性及靈敏性。外周生物標(biāo)志物的分析基于經(jīng)典方法或新的超靈敏技術(shù)，包括ELISA、單分子陣列（SIMOA）、免疫沉淀/MS、液相色譜-MS、免疫磁性還原（IMR）、多聚體檢測(cè)系統(tǒng)、還原石墨烯氧化場(chǎng)效應(yīng)晶體管和冷凍電子顯微鏡。不同的生物標(biāo)志物有其合適的檢測(cè)方法。但是，更可靠、方便、準(zhǔn)確的檢測(cè)手段還需要進(jìn)一步研究探索。
總結(jié)與展望
越來(lái)越多的生物標(biāo)志物運(yùn)用于AD的臨床診斷，提高了AD診斷的準(zhǔn)確性，為AD的早期預(yù)防及早期診斷提供了可能。然而，我們需要進(jìn)一步探索更為穩(wěn)定可靠、特異性高的生物標(biāo)志物及其檢測(cè)方法。同時(shí)，基于A-T-N-X框架的臨床診斷體系也需要進(jìn)一步完善。我們認(rèn)為，需要建立以生物標(biāo)志物為核心，從患者基本信息（包括一般情況）、臨床表現(xiàn)（尤其是認(rèn)知評(píng)估）、基礎(chǔ)檢查及實(shí)驗(yàn)室檢查、基因檢測(cè)等多個(gè)維度全面評(píng)估的AD綜合診斷模型。AD的A-T-N-X框架為研究者提供了一種共同語(yǔ)言。今后應(yīng)更加重視外周生物體液A-T-N-X框架的研究，尤其是利用超靈敏技術(shù)提高外周生物體液Aβ檢測(cè)的準(zhǔn)確性。
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