蔣卓遠，查艷，石小峰，張永彪2,

綜 述

神經(jīng)嵴細胞和神經(jīng)嵴病及其致病機制的研究進展

蔣卓遠1，查艷1，石小峰3,4，張永彪2,3,4

1. 北京航空航天大學生物與醫(yī)學工程學院，北京 100000 2. 北京航空航天大學醫(yī)學科學與工程學院，北京 100000 3. 北京航空航天大學大數(shù)據(jù)精準醫(yī)療高精尖創(chuàng)新中心，北京 100000 4. 工信部大數(shù)據(jù)精準醫(yī)療重點實驗室，北京 100000

神經(jīng)嵴細胞(neural crest cells，NCCs)是一類脊椎動物特有的可遷移的多能干細胞，其可分化為軟骨細胞、神經(jīng)元和黑色素細胞等多種類型細胞。NCCs的形成、遷移和分化受到嚴格調(diào)控，任何擾亂NCCs發(fā)育的因素都可導致胚胎發(fā)育畸形。由神經(jīng)嵴細胞發(fā)育異常所導致的一系列疾病統(tǒng)稱為神經(jīng)嵴病(neurocristopathies，NCPs)。NCPs種類繁多且表型復雜，可累及人體多個部位(顱面部、心臟、腸胃和皮膚等)，嚴重危害患者的身體機能和心理健康。NCPs占所有出生缺陷患兒的1/3，遺傳因素是導致NCPs的主要風險因素，但環(huán)境風險因子以及基因–環(huán)境交互作用異常也可導致NCPs。本文對神經(jīng)嵴細胞和神經(jīng)嵴病及其致病機制進行綜述，為系統(tǒng)認知神經(jīng)嵴細胞發(fā)育以及神經(jīng)嵴病提供參考，為了解神經(jīng)嵴病的病因以及開展有效防控提供科學支撐。

神經(jīng)嵴細胞；神經(jīng)嵴病；基因調(diào)控網(wǎng)絡(luò)；風險因素；發(fā)病機制

神經(jīng)嵴細胞(neural crest cells，NCCs)是脊椎動物胚胎發(fā)育早期的一種具備全胚胎遷移性的全能性干細胞[1]。NCCs經(jīng)過了從軀干向頭部逐步特化的過程，對脊椎動物頭顱的演化起到至關(guān)重要的作用[2,3]。NCCs賦予了脊椎動物高級的感官系統(tǒng)、復雜且精細的神經(jīng)系統(tǒng)以及強有力的掠食器官，支持脊椎動物從被動的濾食行為演化到主動捕食的生活方式[4]。NCCs起源于背側(cè)外胚層的神經(jīng)板邊界，神經(jīng)褶皺的融合誘導了神經(jīng)管的閉合以及NCCs的形成。隨后，神經(jīng)嵴細胞會有序地從胚胎背側(cè)向腹側(cè)遷移，在遷移過程中以二元命運決定方式分化成為祖細胞和間質(zhì)細胞，進而繼續(xù)二元命運決定，分化為軟骨細胞、色素細胞、平滑肌細胞和神經(jīng)元等[5,6]。除此之外，遷移的NCCs還在胚胎發(fā)育早期替代未發(fā)育的吞噬類細胞發(fā)揮死細胞的清除功能[7]。NCCs雖然來自外胚層，但其仍具備分化為中胚層和內(nèi)胚層的能力，因此，有學者將神經(jīng)嵴稱為“第四胚層”，甚至其多能性特征高于其他三個胚層[8]。

NCCs賦予脊椎動物演化優(yōu)勢的同時，也引入了發(fā)育畸形風險。NCCs的發(fā)育過程始終受基因嚴格調(diào)控，任何異常調(diào)控都將擾亂NCCs正常發(fā)育，進而導致胚胎發(fā)育畸形。1974年，Bolande等[9]統(tǒng)梳理了NCCs衍生物異常發(fā)育的系列疾病，并統(tǒng)稱為神經(jīng)嵴病(neurocristopathies，NCPs)。已知的顱面部發(fā)育缺陷大多數(shù)和神經(jīng)嵴細胞發(fā)育相關(guān)，而顱面部出生缺陷患者幾乎占所有人類出生缺陷的1/3[10]，他們臨床癥狀復雜，病因大多未知，分類較為混亂。導致該現(xiàn)狀的主要原因有：(1)NCPs種類繁多，多為散發(fā)罕見病例，樣本收集困難，導致難以進行系統(tǒng)研究；(2)不同NCPs臨床特征紛亂交疊，NCPs的病例癥狀多樣、表型冗雜交疊，目前未形成系統(tǒng)有效的分類體系；(3)NCPs病因缺乏系統(tǒng)揭示，NCPs基本都是獨立研究，如典型代表——唇腭裂和先天性巨結(jié)腸(Hirschsprung)，但在NCCs層面系統(tǒng)的研究各類NCPs的工作仍未開展[11]。本文依據(jù)NCPs的受累器官對其進行系統(tǒng)分類，然后以NCCs的發(fā)育為線索，對NCPs的病因進行梳理，總結(jié)了NCCs發(fā)育異常導致各類NCPs的機制。

1 神經(jīng)嵴細胞

1.1 NCCs的形成

NCCs來源于神經(jīng)板邊界(neural plate border，NPB)內(nèi)的神經(jīng)嵴祖細胞(圖1)。在人類早期胚胎發(fā)育過程中，神經(jīng)嵴祖細胞可誘導促使神經(jīng)板兩側(cè)的NPB隆起，形成神經(jīng)褶皺，隨后隆起的神經(jīng)褶皺融合形成神經(jīng)管，神經(jīng)嵴祖細胞聚集于神經(jīng)管背側(cè)區(qū)域，以等待調(diào)控因子提供特化(specification)信號[1]。

NCCs的形成分為誘導(induction)、特化(specification)和預遷移(pre-migration)三個階段(圖1)。小鼠模型研究揭示NCCs形成過程由多個調(diào)控通路參與，包括骨形態(tài)發(fā)生蛋白(Bmp)信號通路、Wnt信號通路、成纖維細胞生長因子(Fgf)信號通路和Notch信號通路[1]。研究發(fā)現(xiàn)，Bmp信號在NCCs誘導早期開始發(fā)揮作用，然后激活Wnt信號在NCCs的特化階段發(fā)揮調(diào)控活性，他們均對該階段NCCs數(shù)量的維持起關(guān)鍵作用[12]。Fgf信號主要在誘導過程中發(fā)揮作用，F(xiàn)gf8信號的中斷阻止了NPB基因的表達[13]。

1.2 NCCs的遷移

NCCs的遷移始于神經(jīng)管閉合，遷移之初，NCCs經(jīng)上皮–間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition，EMT)過程從神經(jīng)管背側(cè)剝離，然后開始遷移。NCCs的EMT過程是從形成態(tài)向遷移態(tài)的轉(zhuǎn)換，轉(zhuǎn)換后的細胞命運由一系列調(diào)控因子決定。Soldatov等[6]發(fā)現(xiàn)在NCCs的命運決定在遷移時就已發(fā)生，NCCs遷移前階段就分化為兩個不同的亞群：第一是最早的EMT前群，它們由未分層(delamination)的NCCs組成；第二是分層的細胞亞群，其特征在于EMT的關(guān)鍵基因的激活和的沉默，該亞群細胞還被一系列基因的連續(xù)瞬時調(diào)控，如、、和。之后細胞命運決定具體表現(xiàn)為一系列連續(xù)的二元甚至多元決定過程，該過程首先表現(xiàn)為NCCs向感覺類的神經(jīng)膠質(zhì)細胞和其他過渡類型細胞的身份轉(zhuǎn)換，隨后過渡類型細胞再向自主神經(jīng)細胞和間質(zhì)細胞身份轉(zhuǎn)化的二元命運決定過程，這兩類細胞繼續(xù)后續(xù)的命運決定過程，直至分化為最終細胞類型。

圖1 NCCs的發(fā)育過程受信號因子的嚴格調(diào)控

NCCs的形成始于神經(jīng)板邊界，主要由Wnts、Bmps、Fgfs和Notchs信號調(diào)節(jié)。在神經(jīng)管形成早期，神經(jīng)嵴祖細胞位于NPB中，其誘導神經(jīng)板(neural plate，NP)兩側(cè)的NPB隆起形成神經(jīng)褶皺，兩側(cè)的神經(jīng)褶皺融合形成神經(jīng)管后，NCCs便開始誘導–特化–預遷移過程。在大多數(shù)脊椎動物中，NCCs的分層及遷移始于神經(jīng)管閉合，但該過程也可早于神經(jīng)管閉合，比如小鼠的NCCs分層在神經(jīng)管未閉合時即發(fā)生。NCCs：神經(jīng)嵴細胞；NPB：神經(jīng)板邊界；NF：神經(jīng)褶皺；SO：體節(jié)；NT：神經(jīng)管；NO：脊索。

根據(jù)NCCs的形成位置和遷移路線，可將其分為四種類型：顱、軀干、迷走和骶NCCs，遷移后在胚胎的各部位分化成多種功能細胞(圖2：A和B)。顱NCCs從神經(jīng)管背側(cè)遷移出來后，將分裂成三束分散(discrete streams)的細胞流：遷移至中、前腦的背側(cè)細胞流，遷移入鰓弓的腹側(cè)細胞流以及從后腦向其他部位遷移的細胞流[14]。軀干NCCs從第7～28體節(jié)的神經(jīng)管向外遷移的路線有兩條：一條途徑沿背外側(cè)呈連續(xù)狀遷移(continuous wave)，主要發(fā)育為色素細胞；另一條途徑沿腹內(nèi)側(cè)呈分段式(segmented manner)遷移，主要發(fā)育為膠質(zhì)和神經(jīng)元前體[15]。迷走NCCs從菱形腦節(jié)4～7產(chǎn)生后主要遷移到心臟，同時也像骶NCCs遷移到腸道定植，這個是NCCs最長的一條遷移路線[16]。神經(jīng)嵴細胞的遷移受到了多種信號的嚴格調(diào)控[17](圖2C)，包括局部抑制劑，誘導劑和細胞粘附分子，如Snails可直接抑制細胞粘附分子E-cadherin，促進細胞遷移，Eph/ephrin、內(nèi)皮素和Slit/Robo信號決定了NCCs的背外側(cè)和腹內(nèi)側(cè)遷移路徑的選擇。

最新研究發(fā)現(xiàn)NCCs的遷移行為還和外周環(huán)境存在交互，該過程賦予NCCs在早期胚胎發(fā)育中發(fā)揮特殊的功能：在特異吞噬細胞出現(xiàn)之前，遷移的NCCs被證實能在神經(jīng)系統(tǒng)中承擔吞噬細胞碎片的功能。該吞噬過程與巨噬細胞吞噬相似，均由白細胞介素-1所介導[7]。現(xiàn)階段，NCCs在遷移過程中的時空調(diào)控網(wǎng)絡(luò)以及與周圍細胞的互作關(guān)系還亟待深入研究，明晰其遷移轉(zhuǎn)折點的決策機制也將成為后續(xù)研究熱點。

1.3 NCCs的分化

NCCs最終分化成何種細胞和解剖結(jié)構(gòu)類型，主要取決于終點微環(huán)境。當NCCs遇到血管或細胞簇

等提供的終止遷移信號時，便開始分化[11]。顱神經(jīng)嵴細胞(cranial neural crest cells，CNCCs)最終遷移至頭部和咽弓，參與顱面部的骨骼，皮膚，神經(jīng)和肌肉的形成。其他部位的NCCs最終分化三個為：迷走神經(jīng)嵴細胞參與主動脈肺隔、心神經(jīng)節(jié)和腸神經(jīng)節(jié)的形成；軀干神經(jīng)嵴細胞發(fā)育為黑色素細胞、神經(jīng)元、神經(jīng)膠質(zhì)和施旺細胞；骶神經(jīng)嵴細胞分化成交感神經(jīng)節(jié)和腸神經(jīng)節(jié)[11,18](圖2：B和D)。

2 神經(jīng)嵴病

2.1 神經(jīng)嵴病的分類

1974年，Bolande等[9]將由NCCs及其衍生物異常發(fā)育引起的一系列疾病統(tǒng)稱為NCPs，如Treacher Collin綜合征、唇腭裂、Waardenburg綜合征、先天性巨結(jié)腸。隨著研究的深入，那些由于核糖體或纖毛等具體結(jié)構(gòu)發(fā)育異常進而導致NCCs發(fā)育異常的疾病也被列入NCPs范疇，如核糖體病、纖毛病等。然而由于神經(jīng)嵴細胞參與了機體大量細胞和器官的發(fā)育，從而導致神經(jīng)嵴病數(shù)量眾多，加上NCPs表型多樣以及病因認知不足導致神經(jīng)嵴病未被系統(tǒng)梳理。1977年，Reed等[19]依照是否伴發(fā)腫瘤將NCPs劃分為發(fā)育異常(dysplasias)、腫瘤(neoplasms)以及畸形和腫瘤并存的三種類型。2018年，Vega-Lopea等[11]按照神經(jīng)嵴細胞的來源將神經(jīng)嵴病分為顱神經(jīng)嵴病、軀干神經(jīng)嵴病、迷走神經(jīng)嵴病和骶神經(jīng)嵴病。但上述分類方法過于簡單，不利于對NCPs的系統(tǒng)認知。本文將按照神經(jīng)嵴病的主要累及器官，對已知的神經(jīng)嵴病進行分類，同時將和NCCs相關(guān)的腫瘤單獨舉出為一類(圖3)。

圖2 NCCs的遷移和分化

A：NCCs在胚胎中的遷移路徑。NCCs分為顱神經(jīng)嵴、迷走神經(jīng)嵴、軀干神經(jīng)嵴和骶神經(jīng)嵴四個類型。箭頭表示NCCs的遷移流。B：NCCs可分化的組成機體的主要細胞類型。C：NCCs遷移過程的調(diào)控因子和信號通路。D：調(diào)控NCCs分化為各種細胞類型的關(guān)鍵調(diào)控信號。Ⅰ、Ⅱ：第一/二咽弓；OV：聽囊。

2.2 神經(jīng)嵴病的典型病癥

神經(jīng)嵴病可發(fā)生在全身多個部位，或累及單個器官，或同時累及多個器官，形成復雜的神經(jīng)嵴疾病。本文根據(jù)人體的解剖結(jié)構(gòu)進行劃分，總結(jié)了NCPs的典型表型，列舉了NCPs引起的顱面、臟器、四肢、皮膚及全身發(fā)育的異常[20](表1)。顱面部畸形是最常見的典型特征，通常包括上、下頜不完全發(fā)育，牙齒形態(tài)異常，進食和說話困難。下頜的嚴重發(fā)育不良會使嬰幼兒的氣管受到壓迫，氣管狹窄，從而影響呼吸，甚至導致死亡。部分患者眼睛歪斜、睫毛稀疏、眼睛色素異常，導致視力下降。耳朵發(fā)育異常也是多種NCPs的典型特征，如外耳發(fā)育不全或缺失(小耳或無耳)、耳道閉鎖、聽力喪失等[21]。

圖3 NCCs發(fā)育異常導致的神經(jīng)嵴病所累及的人體器官及系統(tǒng)

按照神經(jīng)嵴病的主要累及器官，對已知的神經(jīng)嵴病進行分類。如唇腭裂、半側(cè)顏面短小等都是具有典型顱面部癥狀的神經(jīng)嵴病，先天性巨結(jié)腸是典型的消化道NCP。CDHS：顱面–耳聾–手綜合征(craniofacial-deafness-hand syndrome)；SHFM4：裂手/裂腳畸形4型(split-hand/split-foot malformation type 4)；LMS：四肢-乳腺綜合征(limb-mammary syndrome)；TDO：毛齒骨綜合征(tricho-dento-osseous syndrome)；VHL：Von Hippel-Lindau綜合征(Von Hippel-Lindau syndrome)。

2.3 神經(jīng)嵴病流行病學分析

神經(jīng)嵴病占所有先天性出生缺陷患兒的1/3[10]。據(jù)統(tǒng)計，頜面部第一高發(fā)出生缺陷——唇腭裂發(fā)病率為1/500～1/1000[27]，而頜面部受累器官最多的半側(cè)顏面短小畸形的發(fā)生率在1/5600～1/6550[22]。1歲以下嬰兒最常見的癌癥——神經(jīng)母細胞瘤發(fā)病率為每1/10萬新生兒。消化道典型的神經(jīng)嵴病——先天性巨結(jié)腸(Hirschsprung disease，HSCR)的發(fā)病率約為1/5000新生兒[11]。然而，神經(jīng)嵴病的發(fā)病率很可能被低估了，原因在于醫(yī)生對NCPs的本身及其變異性的認識不足，輕微表型畸形得不到重視，以及疾病無法得到充分且精確診斷。NCPs發(fā)病部位的一個典型特征是存在機體的不對稱性，表現(xiàn)為大部分受累器官僅出現(xiàn)在單側(cè)(表2)。

2.4 神經(jīng)嵴病的防治

當前對于遺傳疾病，以防控為主，診療為輔。由于大量神經(jīng)嵴病患者頜面部畸形明顯，不僅給患者造成身心傷害，后期的矯正性治療還將給家庭和社會帶來巨大經(jīng)濟壓力。將防控關(guān)口前移是出生缺陷防治的重要方向，對存在遺傳風險家族史的孕婦采取產(chǎn)前檢測可有效避免畸形胎兒出生。而在孕期進行適當?shù)姆揽毓芾?，如補充葉酸，避免暴露在致畸劑，如酒精、尼古丁、維甲酸、甲醛等是NCPs防控的較優(yōu)策略。

表1 神經(jīng)嵴病發(fā)病部位和臨床表型

TCS：Treacher Collin綜合征(Treacher Collins syndrome)；CS：顱縫早閉(craniosynostosis)；HFM：半側(cè)顏面短小畸形(hemifacial microsomia)；DBA：先天性再生障礙性貧血(diamond blackfan anemia)；CADSIL：大腦常染色體顯性動脈病變伴皮層下梗死和白質(zhì)腦病(cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)；PHACES：后窩畸形–血管瘤–動脈異常–心臟缺陷–眼睛異常–胸骨裂和臍上中縫(posterior fossa malformations–hemangiomas–arterial anomalies–cardiac defects–eye abnormalities–sternal cleft and supraumbilical raphe)；HSCR：先天巨結(jié)腸(Hirschsprung disease)；AAA：三聯(lián)A綜合征(achalasia- addisonianism-alacrima)：CCHS：先天性中樞性通氣不足癥候群(congenital central hypoventilation)；NAS：Nager 綜合征(nager syndrome)；NS：努南綜合征(noonan syndrome)；OCA：眼–皮膚白化病(oculocutaneous albinism)；HED：少汗性外胚層發(fā)育不良(hypohidrotic ectodermal dysplasia；CPHD：垂體激素缺乏癥(combined pituitary hormone deficiency)；GDFD：發(fā)育遲緩，面部畸形(growth retardation, developmental delay, and facial dysmorphism)。

表2 代表性神經(jīng)嵴病的發(fā)生率及患側(cè)發(fā)生比例

體表畸形的神經(jīng)嵴病的治療主要運用手術(shù)矯正，如唇腭裂、小耳畸形等。NCPs相關(guān)腫瘤的治療也已實現(xiàn)靶向藥物治療和低劑量化療，如神經(jīng)母細胞瘤。體外協(xié)助治療也是行之有效的手段，對于聽力喪失和呼吸困難的患者采取助聽器和通氣治療，可極大改善患者生活質(zhì)量甚至挽救患者生命。隨著科技的不斷進步，基因治療和干細胞治療未來可期：干預基因進而矯正NCCs在TCS患者中的異常發(fā)育已經(jīng)在小鼠模型上獲得成功[31]；母體抗氧化劑修復DNA損傷防止NCCs細胞凋亡可能成為預防神經(jīng)嵴病的有效手段[32]；利用干細胞再生技術(shù)實現(xiàn)受損或畸形的組織修復將是未來NCPs治療研究的重心。

3 神經(jīng)嵴病的病因?qū)W

眾多轉(zhuǎn)錄因子和信號分子協(xié)同調(diào)控了神經(jīng)嵴細胞的復雜發(fā)育過程。在該過程中，任何調(diào)控異常都會擾亂NCCs的形成、遷移和分化，導致NCPs的產(chǎn)生?，F(xiàn)在公認的神經(jīng)嵴病的致病因素是：NCCs基因調(diào)控網(wǎng)絡(luò)(genetic regulation network，GRN)內(nèi)的基因突變、表觀修飾異常以及環(huán)境致畸因素。由于NCPs種類眾多，GRN調(diào)控信號復雜，導致GRN的基因突變、NCCs發(fā)育過程和神經(jīng)嵴病三者的關(guān)系難以系統(tǒng)總結(jié)。下面根據(jù)NCCs發(fā)育過程中基因發(fā)揮調(diào)控活性的時序(附圖1)，梳理了神經(jīng)嵴病的發(fā)病機制。

3.1 基因調(diào)控網(wǎng)絡(luò)異常

3.1.1 NCCs形成異常所致的神經(jīng)嵴病

NCCs的形成是一個多步驟的過程。在NCCs形成前后，、和等神經(jīng)板邊界調(diào)控基因的開始表達，緊隨其后的是更具特異性的NCCs調(diào)控基因，包括、和等[33,34]。其中作為轉(zhuǎn)錄因子的SOX9、PAX3、TFAP2A、FOXD3等信號異常，會影響NCCs的形成過程，導致細胞分裂和細胞的凋亡異常(表3)。在神經(jīng)嵴細胞的生長和增殖過程中，核糖體扮演著重要的角色。核糖體會將信使RNA(mRNA)翻譯成氨基酸序列，參與細胞內(nèi)蛋白的合成。本文以NCCs形成異常所導致的典型神經(jīng)嵴病——核糖體病為例，闡述其發(fā)病機制。

核糖體的形成過程如下：在多種轉(zhuǎn)錄因子的調(diào)控下，首先核糖體DNA(rDNA)會被轉(zhuǎn)錄成pre- rRNA。pre-rRNA生成后，會生成一個中間過渡的轉(zhuǎn)錄本，然后再被加工成成熟的rRNA。RPs與rRNA組裝成核糖體的小亞基(40S)和大亞基(60S)，最后會被運輸?shù)郊毎|(zhì)中進一步形成成熟的核糖體。該過程中，某些基因的突變會造成核糖體的合成異常導致NCPs，其致病機制是因核糖體的合成不足，最終導致NCCs分裂異?；蚣毎蛲龀绦蚴Э兀瑢е翹CCs形成不足[35]。由于相關(guān)基因突變導致核糖體合成異常而影響NCCs形成所產(chǎn)生的NCPs，稱為核糖體病，它是NCPs的一個重要的亞型[36](圖4)。

3.1.2 NCCs遷移異常所致的神經(jīng)嵴病

神經(jīng)嵴細胞的遷移過程由細胞內(nèi)的自主信號以及在遷移過程中與細胞相互作用的周圍環(huán)境共同協(xié)調(diào)調(diào)控[14,17,56,57]。在分層階段，Snails轉(zhuǎn)錄因子家族發(fā)揮了主要的作用。、等基因也是NCCs遷移所必需的，其突變被證實會影響哺乳動物NCCs的分層和遷移。斑駁病、家族性自主神經(jīng)障礙(FD)和II型瓦登伯格綜合征(WSⅡ)等都是典型的由于NCCs遷移異常所導致的神經(jīng)嵴病(表3)[31～33]。

迷走和骶NCCs會遷移到腸道，在腸道形成腸道神經(jīng)元，組成腸道神經(jīng)系統(tǒng)(enteric nervous system，ENS)。許多營養(yǎng)因子、細胞表面受體、轉(zhuǎn)錄因子和信號分子參與了此遷移活動，如Eph/Ephrin、BMP、RET信號等。其中RET是一種跨膜蛋白，受到BMP信號調(diào)控，參與ENS的存活，增殖，遷移和分化過程。EDNRB與RET信號協(xié)同激活MAKP和PI3K通路下游信號和調(diào)控基因，如和基因突變會影響ENS的發(fā)育，導致先天性巨結(jié)腸癥。先天性巨結(jié)腸的主要病因是由于ENS前體細胞不能定植胎兒腸遠端，導致腸道神經(jīng)元缺失，出現(xiàn)腸梗阻(圖5)。

表3 基因調(diào)控網(wǎng)絡(luò)異常導致的神經(jīng)嵴病

PA1/2：第1/2鰓弓；PRS：皮埃爾·羅賓序列(Pierre Robin sequence)；WS：瓦登伯格綜合征(Waardenburg syndrome)；CDHS：顱面–耳聾–手綜合征(craniofacial-deafness-hand syndrome)；BOFS：鰓–眼–面部綜合征(branchio-oculo-facial syndrome)；TCS：Treacher Collins綜合征(Treacher Collins syndrome)；DBA：先天性再生障礙性貧血(diamond blackfan anemia)；MS：米勒綜合征(Miller syndrome)；NS：努南綜合征(noonan syndrome)；HSCR：先天性巨結(jié)腸癥(Hirschsprung disease)；BBB：Opitz G/BBB綜合征(opitz g/bbb syndrome)；FD：家族性自主神經(jīng)障礙(familial dysautonomia)；NB：成神經(jīng)細胞瘤(neuroblastoma)；Tietz：Tietz綜合征(Tietz syndrome)；CADSIL：常染色體顯性遺傳病合并皮質(zhì)下梗死和白質(zhì)腦病(cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)；HH：性腺功能減退癥(hypogonadotropic hypogonadism)；KS：Kallmann綜合征(Kallmann Syndrome)；CPHD：合并垂體激素缺乏(combined pituitary hormone deficiency)；AEC：AEC綜合征(aec syndrome)；LMS：四肢–乳腺綜合征(limb-mammary syndrome)。

3.1.3 NCCs分化異常所致的神經(jīng)嵴病

神經(jīng)嵴細胞的分化是細胞命運決定的結(jié)果，一些轉(zhuǎn)錄因子定義了NCCs的初始分裂狀態(tài)，并賦予了它向不同的譜系分化的潛力。在NCCs遷移過程中，和就已經(jīng)參與了NCCs的命運決定：在NCCs發(fā)育過程的初期開始表達，它決定了NCCs向間充質(zhì)細胞(骨、軟骨和結(jié)締組織)分化的潛能，而則定義了早期的感覺神經(jīng)NCCs祖細胞，將它們和色素NCCs祖細胞進行區(qū)分[6]。之后再通過各類特異性因子調(diào)控，NCCs得以分化形成不同類型的細胞，如PHOX2B與神經(jīng)元的形成相關(guān)、MITF與黑色素細胞發(fā)育相關(guān)、FGF9與嗅神經(jīng)發(fā)育相關(guān)和NOTCH3與血管平滑肌細胞相關(guān)(表3)。

黑色素細胞是NCCs的重要衍生細胞之一，參與了皮膚色素、視網(wǎng)膜色素的形成，還參與內(nèi)耳的發(fā)育，影響胎兒聽力。編碼黑素細胞誘導轉(zhuǎn)錄因子MITF，控制黑色素細胞發(fā)育及其功能。MITF還受到其他NCCs特異基因的調(diào)控：PAX3與SOX10能反式激活其啟動子，協(xié)同上調(diào)的表達[58]。腸道神經(jīng)元分化的調(diào)控信號和黑色素細胞的調(diào)控網(wǎng)絡(luò)存在交互作用，、和除了在NCCs來源的黑色素細胞表達外，也是腸神經(jīng)元細胞增殖、遷移和生存的關(guān)鍵基因。以上幾種基因中的任何突變都會導致皮膚黑色素細胞和腸道中神經(jīng)元的缺失，引起WS和斑駁病(圖6)。

圖4 核糖體的合成異常導致的神經(jīng)嵴病的發(fā)病機制

與NCPs相關(guān)的基因突變會導致核糖體的合成異常，造成顱面部畸形。RNA聚合酶I和III參與rDNA轉(zhuǎn)錄，Treacher Collin綜合征中、和的突變會破壞轉(zhuǎn)錄復合物的形成，影響rRNA的產(chǎn)生。尿嘧啶也參與rRNA的合成，Miller綜合征中的突變干擾尿嘧啶的合成。在RNA聚合酶I和III合成pre-rRNA后，基因(、等)突變破壞核糖體的合成，導致DBA。rDNA：核糖體DNA；pre-rRNA：前核糖體RNA。

圖5 周圍神經(jīng)細胞異常神經(jīng)嵴病的發(fā)病機制

RET是一種跨膜酪氨酸蛋白，RET信號功能涉及ENS的存活、增殖、遷移和分化。RET也受到BMP信號調(diào)控，影響ENS的遷移。RET和EDNRB激活MAKP PI3K通路下游信號：與腸神經(jīng)發(fā)育相關(guān)的PAX3、SOX10和PHOX2B協(xié)同激活RET轉(zhuǎn)錄。和突變也將影響ENS的發(fā)育，導致先天性巨結(jié)腸。Hirschsprung disease：先天巨結(jié)腸癥；Mowat-Wilson syndrome：Mowat-Wilson綜合征。

3.2 表觀遺傳因素

表觀遺傳學即DNA和RNA等大分子的修飾，在NCCs的發(fā)育中也起著重要的調(diào)控作用。表觀遺傳機制是微調(diào)基因表達和細胞分化的關(guān)鍵因素，它將影響基因表達并傳遞給后代。探索NCCs的表觀遺傳學機制，有助于了解NCPs的病因[59]。根據(jù)表觀修飾類型的不同，表4總結(jié)了涉及突變影響的修飾基團、靶基因以及NCCs發(fā)育階段，納入了最新發(fā)現(xiàn)的microRNA的調(diào)控異常導致的NCPs，梳理了不同表觀遺傳修飾異常所導致的神經(jīng)嵴病。

3.3 環(huán)境因素

神經(jīng)嵴病的病因可歸于內(nèi)在遺傳因素和外在環(huán)境因素的異常導致胚胎發(fā)育畸形[80]。研究發(fā)現(xiàn)孕婦能通過食物、飲用水、藥物和空氣傳播以及皮膚接觸到有害的環(huán)境因素，這些都將影響NCCs的發(fā)育過程。據(jù)估計，大約10%～15%的先天出生類缺陷是由于環(huán)境因素(致畸劑)對胚胎發(fā)育的不利影響所導致的[81]。另外，環(huán)境危險因素還可能通過與遺傳因素的相互作用影響NCCs的發(fā)育，如Xu等[22]通過基因–環(huán)境交互作用檢驗研究半側(cè)顏面短小(hemifacial microsomia，HFM)風險因素時，發(fā)現(xiàn)妊娠期父親吸煙與HFM相關(guān)SNP(單核苷酸多態(tài)性，single nucleotide polymorphism)存在顯著交互作用，提示吸煙暴露會增加CFM風險。另外大量的分子、藥物和致畸劑或胎兒毒性物質(zhì)也會影響NCCs的發(fā)育：高糖、維甲酸、四氫大麻酚、可卡因、嗎啡、氯胺酮、血管緊張素轉(zhuǎn)換酶抑制劑、波生坦、氯霉素、卡馬西平、氟康唑、草甘膦除草劑、鋰化合物、甲氨蝶呤、苯巴比妥、他莫昔芬、沙利度胺、甲苯、曲妥珠單抗、維甲酸、丙戊酸等[81]。下面簡要描述一些典型的導致NCCs發(fā)育異常的環(huán)境因素的致畸源作為參考。

圖6 黑色素細胞發(fā)育異常神經(jīng)嵴病

EDNRB和KIT傳導的信號激活MAPK通路，之后作用于MITF的M亞型，該亞型只在黑色素細胞譜系中表達?？刂坪谏丶毎陌l(fā)育和細胞功能。PAX3與SOX10反式激活MITF啟動子，協(xié)同上調(diào)MITF的表達。作為MITF的一種效應(yīng)基因，是Kit和E-鈣粘蛋白基因的轉(zhuǎn)錄抑制因子，影響黑色素細胞的發(fā)育。Piebaldism：斑駁??；Tietz：Tietz綜合征(Tietz syndrome)；WS：Waardenburg綜合征(Waardenburg syndrome)。

表4 表觀遺傳修飾對神經(jīng)嵴發(fā)育的調(diào)控

BDMR：非綜合征性口唇裂及短趾性智力發(fā)育遲滯綜合征(non-syndromic oral clefts and brachydac-tyly mental retardation syndrome)；BOFS：鰓–眼–面部綜合征(branchio-oculo-facial syndrome)；HSCR：先天性巨結(jié)腸癥(Hirschsprung disease)。

3.3.1 乙醇：胎兒酒精譜系障礙(fetal alcohol spectrum disorder，F(xiàn)ASD)

懷孕期間飲酒現(xiàn)在被認為是一種潛在的致畸風險，可能導致以眼瞼裂隙短上唇延長和發(fā)育不全為特征的胎兒酒精譜系障礙[82]。懷孕期間過度接觸酒精會導致胎兒顱面畸形，占所有酒精導致的出生缺陷的1/3。研究發(fā)現(xiàn)將早期胚胎暴露在乙醇中，發(fā)現(xiàn)細胞內(nèi)的酶活性降低，核糖體凋亡，并破壞了神經(jīng)的發(fā)育，其中包括神經(jīng)元存活、增殖、細胞遷移和突觸的形成都受到了影響[83]。取雞胚胎研究的額外證據(jù)表明，酒精能抑制Wnt和Shh信號，從而改變NCCs的遷移和存活[84]。最終導致顱和心臟NCCs數(shù)量減少，遷移不流足，引發(fā)顱面部畸形甚至先天性心臟病[81]。

3.3.2 維甲酸(retinoic acid，RA)

維生素A胚胎病是一種由子宮內(nèi)過度暴露于維甲酸引起的NCP，以小耳朵、頜骨發(fā)育不全、腭裂和心臟異常為特征，RA已被證明對NCCs的形成至關(guān)重要[85,86]。RA攝入量的細微差異都將對NCCs的發(fā)展產(chǎn)生重大影響，孕期服用RA藥物會損害正常的顱面形態(tài)發(fā)生。過量的RA也會影響涉及顱骨發(fā)育的的表觀遺傳效應(yīng)。RA還被發(fā)現(xiàn)會導致小鼠的口腔–面部骨折，與兩個同源框基因2和表達異常相關(guān)[87]。此外，RA信號的改變可導致一種嚴重的致盲疾?。合忍煨郧喙庋踇88]?？梢钥隙ǖ氖荝A致畸的分子基礎(chǔ)是在胚胎期RA刺激p53過度激活，增加了NCCs的凋亡，導致顱骨、心臟甚至骶骨NCCs缺乏[89,90]。

3.3.3 煙霧：香煙、尼古丁、煙草、煙塵

抽煙(尼古丁)也是導致出生類缺陷的重要風險因素，孕婦孕期接觸香煙會導致胎兒煙草綜合征(fetal tobacco syndrome，F(xiàn)TS)，除了呼吸異常和心臟缺陷外，F(xiàn)TS的主要特征是唇腭裂[91]。在非洲爪蟾胚胎和哺乳動物NCCs中進行的一項研究表明，尼古丁和霧化電子煙液的暴露會導致多種出生缺陷，包括正中面部裂隙和中面部發(fā)育不全[92]。胚胎暴露在香煙煙霧提取物(一種含有低水平的芳香烴受體(aromatic hydrocarbon receptor，AHR)配體化合物)中，主要通過下調(diào)Wnt信號通路的共激活因子R-spondin1影響Wnt通路，該通路是遷移性NCCs的關(guān)鍵信號[93]。皮膚中的所有細胞都表達AHR，如在黑素細胞、朗格漢斯細胞(langerhans cells，LCs)、表皮外層和成纖維細胞等[94]。因此長期暴露在尼古丁下的胎兒，心臟、皮膚等發(fā)育都將受到影響。

3.3.4 葉酸(folic acid, FA)：甲氨蝶呤(metho-trexate, MTX)

葉酸對神經(jīng)嵴細胞的發(fā)育至關(guān)重要。在懷孕期，合理的葉酸補充可以防止神經(jīng)管缺陷、顱面畸形和心臟缺陷的發(fā)生[95]。此外，體外和體內(nèi)實驗表明，葉酸水平的異常變化可導致雞心NCCs的異常遷移和分化。在早期的NCCs特化過程中，正常的口腔發(fā)育需要DHFR信號[96]。MTX是FA的拮抗劑，它競爭性地抑制二氫葉酸還原酶(dihydrofolate reductase，DHFR)，導致葉酸的缺乏[96,97]。在雞胚實驗中，MTX處理會促使兩種主要的葉酸轉(zhuǎn)運蛋白FolR1和RFC1的下調(diào)，影響和的正常表達，擾亂了口腔面部組織的形成，推測葉酸可能參與了胎兒口面組織的發(fā)育[98]。

4 結(jié)語與展望

神經(jīng)嵴細胞對脊椎動物頭部的“精細雕琢”賦予其進化上的絕對優(yōu)勢。在人體內(nèi)，其形成了復雜有序的神經(jīng)系統(tǒng)與感觀系統(tǒng)，使人類擁有強大的認知和創(chuàng)造能力。然而，當今人們對神經(jīng)嵴細胞的認知依舊較淺，雖然現(xiàn)有研究對NCCs的形成、遷移和分化進行了解析，并在組織和單細胞維度初步揭示了NCCs多能性機制、細胞命運決定模式和發(fā)育進程，但NCCs的發(fā)育和命運如何被精準調(diào)控、NCCs的遷移路線如何決定、NCCs和周圍細胞的如何交互共分化、在時空層面NCCs如何被精準調(diào)控以及基因突變影響了NCCs的哪個發(fā)育階段等科學問題仍有待深入揭示。

由于神經(jīng)嵴細胞發(fā)育過程復雜，受到多種信號因子的調(diào)節(jié)，導致被擾亂的神經(jīng)嵴細胞與最終發(fā)生畸變的組織類型之間匹配困難：即具有相同表型的NCPs可能是由不同NCCs類型的不同發(fā)育過程被擾亂所引起，如先天性巨結(jié)腸既可由NCCs遷移異常也可由分化異常所導致[75]；不同表型的NCPs可能是由同一基因在NCCs不同發(fā)育階段的突變導致的，如SOX10的突變可以在NCCs遷移或分化中分別導致先天性巨結(jié)腸或WS[99]。因此神經(jīng)嵴病的癥狀具有較大的表型異質(zhì)性和病因同源性，這給研究NCCs發(fā)育過程和NCPs的致病機制帶來了嚴峻考驗。精準明晰NCPs的致病突變將有助于實施針對性的治療和預防手段[100]，在基因、蛋白和功能層面的在體外動物實驗有助于闡明NCPs的致病機制，并揭示關(guān)鍵治療靶點[101]。

隨著單細胞RNA測序的進展，特別是單細胞多組學方法的快速發(fā)展，神經(jīng)嵴譜系中各類細胞的形成節(jié)點和驅(qū)動其多能性的機制也逐漸被解析。NCCs單細胞數(shù)據(jù)證實了之前對神經(jīng)嵴基因調(diào)控網(wǎng)絡(luò)的認知，并且揭示了許多新的轉(zhuǎn)錄調(diào)控因子，并發(fā)現(xiàn)了新的調(diào)控網(wǎng)絡(luò)(迷走–神經(jīng)嵴調(diào)控網(wǎng)絡(luò))[102,103]。同時經(jīng)典的神經(jīng)胚層假說也可以驗證：即NCCs起源于神經(jīng)外胚層，具備分化為多種類型細胞的多能性。最新研究揭示NCCs可被Oct4重編程恢復至囊胚期的多能性，進而分化為其他胚層細胞[104]，從而推測NCCs不僅僅是“第四胚層”，其多能性甚至要高于內(nèi)、中和外胚層。Soldatov等[6]利用NCCs的單細胞數(shù)據(jù)發(fā)現(xiàn)NCCs的命運似乎從遷移開始前后就已經(jīng)被決定，NCCs的命運決定有三個主要階段：(1)遷移前后的協(xié)同激活，(2)遷移過程中的命運逐步偏倚和(3)遷移結(jié)束的分化階段不同的細胞決策。然而，單細胞方法不可避免地失去了空間/解剖位置的信息，空間組學的最新進展將在不久的將來揭示神經(jīng)嵴細胞命運如何在正確的時間和地點被激活以形成相應(yīng)的衍生物，為神經(jīng)嵴細胞的命運和功能帶來新的認知，為神經(jīng)嵴病的病因?qū)W揭示和疾病防控及治療提供關(guān)鍵技術(shù)。

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附圖1 NCPs分類統(tǒng)計Venn圖

Supplementary Fig. 1 Venn chart of NCPs classification

根據(jù)受累神經(jīng)嵴細胞發(fā)育過程對神經(jīng)嵴病進行分類，重疊部分清楚地顯示了某些NCPs歸因于多個過程發(fā)育受阻。

Abbreviations of NCPs described in this article: TCS: Treacher Collins syndrome Treacher Collins綜合征；KS: Kallmann Syndrome Kallmann綜合征；PRS: Pierre Robin Sequence皮埃爾·羅賓序列；HPE: holoprosencephaly前腦無裂畸形；HH: hypogonadotropic hypogonadism 性腺功能減退癥；MBS: Moebius Syndrome莫比烏斯綜合征；3MC: 3MC Syndrome 3MC綜合征；ADULT: ADULT Syndrome ADULT綜合征；AEC: AEC Syndrome AEC綜合征；AS: Alagille Syndrome Alagille綜合征；BOR: Branchio-Oto-Renal Syndrome鰓-耳-腎綜合征；CADASIL: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy大腦常染色體顯性動脈病變伴皮層下梗死和白質(zhì)腦病；CDHS: Craniofacial-Deafness-Hand Syndrome 顱面-耳聾-手綜合征；GCMN: Giant Congenital Melanocytic Nevi巨大先天性黑素細胞痣；NF1: Neurofibromatosis I Ⅰ型神經(jīng)纖維瘤；WS: Waardenburg syndrome瓦登伯格綜合征；CED: Cranioectodermal dysplasia/Sensenbrenner syndrome顱骨外胚層發(fā)育不良/Sensenbrenner綜合征；EEC3: EEC3 syndrome EEC3綜合征；EVC: Ellis-van Creveld syndrome軟骨外胚層發(fā)育不良；FASD: Fetal Alcohol Spectrum Disorder胎兒酒精譜系障礙；GDFD: Growth Retardation, Developmental Delay, and Facial Dysmorphism生長遲緩，發(fā)育遲緩，面部畸形；HCS: Hajdu-Cheney Syndrome Hajdu-Cheney綜合征；FS: Klippel-Feil Syndrome先天性短頸綜合征；LMS: Limb-Mammary Syndrome四肢-乳腺綜合征；MFDA: Mandibulofacial Dysostosis with Alopecia頜面部發(fā)育不良伴脫發(fā)；MS: Multiple sclerosis多發(fā)性硬化癥；NAD: Nager Acrofacial Dysostosis Nager綜合征；NS: Noonan syndrome Noonan綜合征；PA: Peters Anomaly Peter異常/前段發(fā)育不良；PHACE: PHACE Syndrome PHACE綜合征；SAMS: SAMS disorder SAMS異常；SHFM4: Split-hand/split-foot malformation type 4裂手/裂腳畸形4型；TDO: Tricho-dento-osseous syndrome毛齒骨綜合征；22q11: 22q11.2 Deletion Syndrome (DGS) 22q11.2缺失綜合癥；AAA: Achalasia- Addisonianism-Alacrima Syndrome AAA綜合征；ACS: Auriculo Condylar Syndrome耳廓-髁狀突綜合征；ARS: Axenfeld- Rieger Syndrome Axenfeld-Rieger綜合征；BLS: Bamforth-Lazarus syndrome Bamforth-Lazarus綜合癥；BOFS: Branchio-Oculo-Facial Syndrome 鰓-眼-面部綜合征；BS: Binder Syndrome Binder綜合征；CCHS: Congenital Central Hypoventilation Syndrome先天性中樞性通氣不足綜合征；CHARGE: CHARGE syndrome CHARGE綜合征；CMT: Charcot-Marie-Tooth and Deafness Syndrome腓骨肌萎縮癥和耳聾綜合癥；CS: Craniosynostosis顱縫早閉；DBA: Diamond Blackfan Anemia先天性再生障礙性貧血；FD: Familial dysautonomia家族性自主神經(jīng)障礙；FMTC: Familial medullary thyroid carcinomas家族性甲狀腺髓樣癌；HFM: Hemifacial microsomia半側(cè)顏面短小.

Research progress on neural crest cells and neurocristopathies and its pathogenesis

Zhuoyuan Jiang1, Yan Zha1, Xiaofeng Shi3,4, Yongbiao Zhang2,3,4

Neural crest cells (NCCs) are multipotent progenitor cells unique to vertebrates, and they have the ability to differentiate into a variety of cells, such as chondrocytes, neurons, and melanocytes. The formation, migration, and differentiation of NCCs are tightly regulated, and the disruption of NCC development results in abnormal embryo development. Neurocristopathies (NCPs) refer to a group of diseases that develop in response to abnormal development of NCCs. NCPs are of various types and exhibit complex phenotypes, which can affect many parts of the human body, such as the craniofacial structure, heart, intestine, and skin. NCPs negatively impact the physical function and mental health of the affected patients. NCPs account for one third of the defects in children with birth defects. Genetic factors are the main risk factors for NCPs, but environmental factors and abnormal gene-environment interactions can also lead to the development of NCPs. In this review, we introduce NCCs, NCPs, and their pathogenesis, so as to provide a reference point for a systematic understanding of NCPs and NCC development, and to provide scientific support for understanding the etiology of NCPs and their effective prevention and control.

neural crest cells (NCCs); neurocristopathy; genetic regulation network; risk factors; pathogenesis

2021-09-29;

2021-12-11;

2022-01-04

國家自然科學基金(編號：82171844，81970898)和北京市自然科學基金(編號：7204273)資助[Supported by the National Natural Science Foundation of China (Nos. 82171844, 81970898) and Beijing Natural Science Foundation Project (No. 7204273)]

蔣卓遠，在讀碩士研究生，專業(yè)方向：基礎(chǔ)醫(yī)學。E-mail: SY1910307@buaa.edu.cn

張永彪，博士，副研究員，研究方向：生物信息學。E-mail: zhangyongbiao@buaa.edu.cn

10.16288/j.yczz.21-253

(責任編委: 何淑君)