郭佳妮，劉帆，王璐

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斑馬魚血液疾病模型及應(yīng)用

郭佳妮，劉帆，王璐

中國(guó)醫(yī)學(xué)科學(xué)院血液病醫(yī)院(中國(guó)醫(yī)學(xué)科學(xué)院血液學(xué)研究所)，實(shí)驗(yàn)血液學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，國(guó)家血液系統(tǒng)疾病臨床醫(yī)學(xué)研究中心，天津 300020

血液發(fā)育是一個(gè)復(fù)雜有序且保守的過(guò)程，由多種轉(zhuǎn)錄因子和信號(hào)通路協(xié)同調(diào)控，任何環(huán)節(jié)的失調(diào)都可能引起血液系統(tǒng)發(fā)育或功能缺陷，導(dǎo)致血液疾病的發(fā)生。斑馬魚()造血過(guò)程及分子調(diào)控機(jī)制與哺乳動(dòng)物高度保守。應(yīng)用斑馬魚模擬致病因子的異常變化構(gòu)建相關(guān)血液疾病模型，為探究疾病發(fā)生機(jī)制、腫瘤發(fā)生發(fā)展可視化研究及高通量化學(xué)篩選提供了有力的工具。本文概述了斑馬魚血液疾病模型及其應(yīng)用，這些疾病模型不僅有助于完善對(duì)血液系統(tǒng)病理生理學(xué)、血液疾病發(fā)生分子機(jī)制的認(rèn)識(shí)，也為臨床相關(guān)惡性血液疾病的治療提供了新思路。

斑馬魚；血液疾病；動(dòng)物模型；化學(xué)藥物篩選

自20世紀(jì)80年代美國(guó)俄勒岡大學(xué)George Streisinger首次使用斑馬魚()作為模式生物以來(lái)[1]，越來(lái)越多的實(shí)驗(yàn)室開始使用斑馬魚進(jìn)行發(fā)育生物學(xué)及人類疾病的相關(guān)研究[2,3]。斑馬魚是一種淡水硬骨魚，具有許多獨(dú)特的生物學(xué)優(yōu)勢(shì)：體外受精與胚胎透明，便于觀察與操作；繁殖能力強(qiáng)且生長(zhǎng)迅速，有利于大規(guī)模篩選等[4]。此外，硬骨魚類和哺乳動(dòng)物擁有相當(dāng)數(shù)量的同源基因，如82%的人類已知致病基因在斑馬魚中存在同源基因[5]，且針對(duì)這些基因構(gòu)建的斑馬魚突變體可作為人類相關(guān)疾病研究的動(dòng)物模型[6,7]。目前，日漸成熟的基因編輯技術(shù)使得斑馬魚疾病模型的構(gòu)建更加簡(jiǎn)便快捷。通過(guò)ZFN (zinc finger nucleases)、TALEN (transcrip-tion activator-like effector nuclease)以及CRISPR/Cas9 (clustered regularly in-terspaced short palindromic repeats/CRISPR associa-ted 9)技術(shù)進(jìn)行基因編輯，科研人員獲取斑馬魚特定基因缺失突變體，進(jìn)行表型及功能的研究[8~10]。中國(guó)科研人員Sun等[11]應(yīng)用CRISPR/Cas9技術(shù)對(duì)斑馬魚1號(hào)染色體基因進(jìn)行敲除，獲得了大量突變體，其中約有1/4突變體可以模擬人類相關(guān)疾病?；谧陨淼莫?dú)特優(yōu)勢(shì)以及先進(jìn)的技術(shù)方法，斑馬魚已逐漸成為研究胚胎發(fā)育與人類疾病發(fā)生機(jī)制的重要模型。

1 斑馬魚血液發(fā)育過(guò)程

血液系統(tǒng)是維持機(jī)體生命活動(dòng)重要的系統(tǒng)之一，為機(jī)體提供氧氣和營(yíng)養(yǎng)物質(zhì)，通過(guò)物質(zhì)交換維持內(nèi)環(huán)境的穩(wěn)態(tài)，同時(shí)提供免疫防御與保護(hù)。血液系統(tǒng)包含紅系細(xì)胞、髓系細(xì)胞以及淋系細(xì)胞等多種成熟血細(xì)胞，而這些血細(xì)胞均由造血過(guò)程產(chǎn)生。造血過(guò)程是造血干細(xì)胞(hematopoietic stem cells, HSCs)及各類血細(xì)胞產(chǎn)生、分化及發(fā)育成熟的過(guò)程，開始于胚胎發(fā)育早期并貫穿整個(gè)生命過(guò)程[12]。

脊椎動(dòng)物造血發(fā)育是高度保守的過(guò)程，從胚胎發(fā)育早期到成體，造血過(guò)程的階段性是一致的，分為初級(jí)造血和次級(jí)造血兩個(gè)階段[13,14]。初級(jí)造血為早期胚胎發(fā)育提供必要的髓系細(xì)胞和紅細(xì)胞。斑馬魚的初級(jí)造血發(fā)生在兩個(gè)解剖位置—前側(cè)板中胚層以及由后側(cè)板中胚層發(fā)育形成的中間細(xì)胞團(tuán)(inter-mediate cell mass, ICM)，分別生成初級(jí)髓系細(xì)胞和初級(jí)紅系細(xì)胞[4]。和等基因?qū)Τ跫?jí)造血與血管的發(fā)生至關(guān)重要[15~17]；和(也被稱為)分別促進(jìn)紅系和髓系的分 化[18,19]。初級(jí)造血是一個(gè)瞬時(shí)過(guò)程，很快被次級(jí)造血過(guò)程替代。次級(jí)造血過(guò)程始于造血干細(xì)胞的產(chǎn)生，位于主動(dòng)脈–性腺–中腎(aorta-gonad-mesenephros, AGM)區(qū)域背主動(dòng)脈腹側(cè)壁的生血內(nèi)皮細(xì)胞，經(jīng)過(guò)內(nèi)皮造血轉(zhuǎn)化過(guò)程生成HSCs[20,21]。隨后HSCs遷移至尾部造血組織(caudal hematopoietic tissue, CHT) (相當(dāng)于哺乳動(dòng)物胎肝)，經(jīng)過(guò)短暫擴(kuò)增之后，大部分HSCs遷移并定植于腎髓(相當(dāng)于哺乳動(dòng)物骨髓)，一部分HSCs則遷移到胸腺分化為T淋巴前體細(xì)胞[22]。HSC命運(yùn)決定的關(guān)鍵基因包括、以及等[23~26]。與初級(jí)造血相似，和驅(qū)動(dòng)紅細(xì)胞生成[15,18]，和促進(jìn)髓系細(xì)胞分化[19,27]。此外，、和等關(guān)鍵基因影響淋系細(xì)胞的分化[28~33]。

脊椎動(dòng)物血液發(fā)育是維持機(jī)體正常生命活動(dòng)的動(dòng)態(tài)過(guò)程，受多種關(guān)鍵因子和信號(hào)通路的精細(xì)協(xié)同調(diào)控，多個(gè)造血組織和器官參與其中。在此過(guò)程中，任何環(huán)節(jié)的失調(diào)都可能引起血液系統(tǒng)發(fā)育或功能缺陷，最終導(dǎo)致血液疾病的發(fā)生。因此系統(tǒng)了解并深入探究血液發(fā)育的調(diào)控機(jī)理有助于解析疾病發(fā)生機(jī)制。

2 斑馬魚血液疾病模型及其應(yīng)用

血液病是原發(fā)于血液系統(tǒng)，或影響造血系統(tǒng)伴發(fā)血液異常的一類疾病。血液病種類繁多，且多數(shù)為難治性或惡性疾患，近年來(lái)發(fā)病率有逐漸增高的趨勢(shì)，其中白血病的發(fā)病率和死亡率更是居于我國(guó)惡性腫瘤前10位。因此，針對(duì)疾病臨床需求深入開展基礎(chǔ)研究，解析疾病發(fā)病機(jī)制具有重要意義。應(yīng)用模式動(dòng)物，針對(duì)關(guān)鍵致病因子構(gòu)建疾病模型，對(duì)于人類疾病機(jī)制研究、治療及藥物評(píng)價(jià)至關(guān)重要。斑馬魚造血過(guò)程及調(diào)控機(jī)制與哺乳動(dòng)物高度保守，結(jié)合體外受精、早期胚胎透明、豐富的轉(zhuǎn)基因魚系等獨(dú)特優(yōu)勢(shì)，斑馬魚已成為造血發(fā)育以及人類疾病研究的常用模式動(dòng)物之一。在斑馬魚體內(nèi)模擬致病因子異常變化構(gòu)建相關(guān)血液疾病模型，可用于腫瘤發(fā)生發(fā)展可視化研究及高通量化學(xué)篩選。本文系統(tǒng)總結(jié)了斑馬魚白血病模型和其他多種血液疾病模型的構(gòu)建，及其在疾病研究中的應(yīng)用。

2.1 白血病模型

白血病是一類造血干祖細(xì)胞惡性克隆性疾病，按細(xì)胞類型和生長(zhǎng)速度主要分為4類：急性淋巴細(xì)胞白血病(acute lymphoblastic leukemia, ALL)、慢性淋巴細(xì)胞白血病(chronic lymphocytic leukemia, CLL)、急性髓系白血病(acute myeloid leukemia, AML)和慢性髓系白血病(chronic myeloid leukemia, CML)。其中ALL可以分為急性T淋巴細(xì)胞白血病(acute T-lymphocytic leukemia, T-ALL)和急性B淋巴細(xì)胞白血病(acute B-lymphocytic leukemia, B-ALL)。

2.1.1 T-ALL模型

T細(xì)胞惡性腫瘤是一類分子異質(zhì)性疾病，由復(fù)雜的遺傳變化所驅(qū)動(dòng)[34]。淋巴細(xì)胞惡性轉(zhuǎn)化的原因主要是異常的染色體易位和異常激活的信號(hào)通路[35,36]。在斑馬魚體內(nèi)模擬這些改變可以構(gòu)建T-ALL相關(guān)轉(zhuǎn)基因斑馬魚系。

原癌基因影響細(xì)胞生長(zhǎng)和增殖，在人類癌癥發(fā)生過(guò)程中發(fā)揮重要作用[37]。第一個(gè)T-ALL轉(zhuǎn)基因斑馬魚模型是應(yīng)用斑馬魚啟動(dòng)子驅(qū)動(dòng)融合基因表達(dá)，借助該模型美國(guó)哈佛大學(xué)Thomas Look團(tuán)隊(duì)首次實(shí)時(shí)觀察EGFP標(biāo)記白血病細(xì)胞的發(fā)生發(fā)展過(guò)程[38]。然而，該模型的大多數(shù)后代在性成熟前便發(fā)展為晚期白血病，無(wú)法自然擴(kuò)繁，需要通過(guò)體外受精進(jìn)行繁殖，操作繁瑣。為了解決這一問(wèn)題，Thomas Look團(tuán)隊(duì)構(gòu)建了條件性轉(zhuǎn)基因魚系Tg ()。正常狀態(tài)下該品系不發(fā)生白血病，Cre表達(dá)后才可能發(fā)生T-ALL，然而誘導(dǎo)率僅有13%[39]。為了突破低疾病外顯率的限制，該團(tuán)隊(duì)引入熱休克啟動(dòng)子驅(qū)動(dòng)表達(dá)。在受精后3天進(jìn)行熱休克處理，81%的轉(zhuǎn)基因魚發(fā)生T淋巴母細(xì)胞性淋巴瘤(T-lymphob-lastic lymphoma, T-LBL)，并快速進(jìn)展為T-ALL，該模型大大提高了誘導(dǎo)T-ALL的效率[40]。近期，Jiang等[41]應(yīng)用此疾病模型證明蛋白激酶Aurora B能夠結(jié)合并磷酸化MYC，形成AURKB-MYC正反饋調(diào)控軸，加快T-ALL發(fā)生發(fā)展。

T-ALL病例中也常見到PTEN/PI3K/Akt通路發(fā)生遺傳改變[42]。Thomas Look團(tuán)隊(duì)構(gòu)建了誘導(dǎo)型T-ALL轉(zhuǎn)基因斑馬魚模型，由4-羥基他莫昔芬誘導(dǎo)，激活原癌基因?qū)е录膊“l(fā)生。撤除4-羥基他莫昔芬引起腫瘤消退，而斑馬魚基因功能缺失突變或持續(xù)激活型Akt2表達(dá)則可促進(jìn)腫瘤繼續(xù)發(fā)展。這些發(fā)現(xiàn)說(shuō)明，即使癌基因驅(qū)動(dòng)的信號(hào)丟失，Akt通路激活也足以維持腫瘤生存[43]。T細(xì)胞前體向T-ALL轉(zhuǎn)化的另一個(gè)關(guān)鍵基因是，約65%的T-ALL患者基因激活[44]。應(yīng)用啟動(dòng)子驅(qū)動(dòng)胞內(nèi)段表達(dá)，成功構(gòu)建了T-ALL模型。同時(shí)證明了Notch激活與抗凋亡分子Bcl2過(guò)表達(dá)相結(jié)合進(jìn)一步加速了T-ALL發(fā)生，提示二者之間的協(xié)同作用[45]。

除了針對(duì)已知遺傳學(xué)改變構(gòu)建疾病模型，表型驅(qū)動(dòng)的正向遺傳篩選方法也得到廣泛應(yīng)用，美國(guó)猶他大學(xué)Nikolaus Trede團(tuán)隊(duì)?wèi)?yīng)用N-乙基-N-亞硝基脲(N-ethyl-N-nitrosourea，ENU)化學(xué)誘變，篩選并鑒定了具有遺傳傾向的3個(gè)T細(xì)胞惡性腫瘤魚系—()、()和()[46]。和突變體作為家族性白血病和淋巴瘤的模型已被多次報(bào)道[47~50]。利用這些突變體進(jìn)行重疊修飾因子篩選(superimposed modifier screens)，識(shí)別影響疾病表型的協(xié)同基因。具有低外顯率和長(zhǎng)潛伏期的雜合突變體適合于篩選腫瘤發(fā)生促進(jìn)因子；高外顯率且早期發(fā)病的純合突變體是篩選腫瘤抑制途徑的最佳選擇。此外，這些魚系的腫瘤臨床癥狀和分子特征與人類T-ALL和T-LBL相似，其中胸腺腫瘤癥狀類似于哺乳動(dòng)物淋巴瘤模型和人類患者中的縱隔腫塊[46]。從胸腺腫瘤、其他部位的播散性克隆或連續(xù)移植后的高度侵襲性腫瘤中分別純化腫瘤細(xì)胞，比較分析疾病進(jìn)展各個(gè)階段的細(xì)胞特征，有助于揭示腫瘤轉(zhuǎn)化的分子事件[46]。這些突變體作為T細(xì)胞惡性腫瘤的理想模型，為該疾病的研究提供了新的工具。

2.1.2 B-ALL模型

染色體易位t(12;21)是兒童B-ALL中最常見的易位形式，其導(dǎo)致的(又稱)融合基因存在于25%的患兒中[51,52]。然而，在小鼠()中建立該融合基因B-ALL模型并不成功[53]。后續(xù)研究中應(yīng)用不同的啟動(dòng)子，建立了泛表達(dá)或淋系祖細(xì)胞特異表達(dá)人的多種斑馬魚模型，但其中只有少數(shù)斑馬魚發(fā)生了B-ALL[54]。近期研究發(fā)現(xiàn)，T-ALL模型Tg()會(huì)同時(shí)發(fā)生B-ALL，借助淋系特異標(biāo)記轉(zhuǎn)基因品系Tg ()，發(fā)現(xiàn)T-ALL細(xì)胞呈現(xiàn)強(qiáng)綠色熒光，而B-ALL細(xì)胞則呈現(xiàn)較弱綠色熒光；此外，與基因分別表達(dá)于B-ALL與T-ALL，可以區(qū)分兩種細(xì)胞簇?；谝陨咸卣?，T-ALL模型Tg()可用于B-ALL疾病的研究[55]。

2.1.3 AML模型

染色體重排后產(chǎn)生的致癌融合基因可驅(qū)動(dòng)急性髓系白血病的發(fā)生，如inv(8)(p11;q13)導(dǎo)致融合基因[56]，t(8;21)q(21;22)導(dǎo)致融合基因[57]，以及t(7;11)(p15;p15)導(dǎo)致()融合基因[58]。在斑馬魚中表達(dá)常見的致癌融合基因可以構(gòu)建AML疾病模型。

最早的AML模型是在斑馬魚胚胎中短暫表達(dá)人融合癌基因，然而這些模型均存在早期致死現(xiàn)象，無(wú)法在成體期進(jìn)行研究[59,60]。第一個(gè)成功的非胚胎致死AML模型是用啟動(dòng)子驅(qū)動(dòng)融合基因表達(dá)，該模型中髓系前體細(xì)胞廣泛侵襲斑馬魚腎臟，然而AML發(fā)病率低、潛伏期長(zhǎng)[61]。在時(shí)間上控制致癌基因的表達(dá)可以有效解決胚胎死亡問(wèn)題。在斑馬魚胚胎中熱激處理誘導(dǎo)融合基因表達(dá)可模擬人AML部分癥狀，機(jī)制研究表明通過(guò)影響紅-髓系祖細(xì)胞分化，促進(jìn)粒細(xì)胞產(chǎn)生。同時(shí)用組蛋白去乙酰酶抑制劑Trichos-tatin A處理可以恢復(fù)和的表達(dá)，引起的粒細(xì)胞聚集也得以改善[62]。應(yīng)用該模型開展的生物活性化合物篩選，結(jié)果顯示環(huán)氧合酶-2 (COX-2)選擇性抑制劑—尼美舒利能拮抗導(dǎo)致的異常造血分化[63]。而將熱激活驅(qū)動(dòng)與Cre/loxP系統(tǒng)結(jié)合，則可以實(shí)現(xiàn)在特定時(shí)間和空間位置誘導(dǎo)癌基因表達(dá)。應(yīng)用轉(zhuǎn)基因魚系Tg ()與Tg ()雜交，可以特異性地在髓系細(xì)胞中誘導(dǎo)致癌基因表達(dá)。胚胎期過(guò)表達(dá)可干擾早期造血發(fā)育，導(dǎo)致髓系前體占優(yōu)勢(shì)；而在成體期過(guò)表達(dá)可導(dǎo)致23%的轉(zhuǎn)基因魚在19~23個(gè)月時(shí)出現(xiàn)骨髓增殖性腫瘤(myeloprolifera-tive neoplasms, MPN)[64]。利用此模型，加拿大戴爾豪斯大學(xué)Jason Berman團(tuán)隊(duì)發(fā)現(xiàn)DNA甲基轉(zhuǎn)移酶抑制劑與組蛋白去乙?；敢种苿┙Y(jié)合可恢復(fù)過(guò)表達(dá)胚胎的正常血液發(fā)育[65]，該發(fā)現(xiàn)揭示了與表觀遺傳調(diào)控之間的聯(lián)系，展示了協(xié)同藥物組合在9誘導(dǎo)的髓系疾病中的治療潛力。

除了染色體重排，基因表達(dá)水平的改變也與AML發(fā)生密切相關(guān)。癌基因()在AML患者體內(nèi)過(guò)度表達(dá)，可作為AML不良預(yù)后的標(biāo)志[66]。Shen等[67]構(gòu)建了由熱休克元件(heat shock elements, HSE)驅(qū)動(dòng)小鼠基因表達(dá)的AML模型。通過(guò)上調(diào)與增強(qiáng)初級(jí)造血，并通過(guò)誘導(dǎo)和促進(jìn)髓系細(xì)胞擴(kuò)增，導(dǎo)致外周血髓系前體細(xì)胞積累。此外，細(xì)胞周期進(jìn)程改變、糖代謝異常、MAPK/Ras及p53信號(hào)途徑均參與導(dǎo)致的血細(xì)胞惡性轉(zhuǎn)化過(guò)程。該模型AML發(fā)病率高且潛伏期短，為研究MYCN致癌作用的分子調(diào)控網(wǎng)絡(luò)建立了有力工具。

2.2 其他血液腫瘤模型

2.2.1 骨髓增殖性腫瘤模型

骨髓增殖性腫瘤(MPN)起源于造血干細(xì)胞，表現(xiàn)為骨髓一系或多系血細(xì)胞過(guò)度增殖，包括真性紅細(xì)胞增多癥(polycythemia vera, PV)、原發(fā)性血小板增多癥(essential thrombocythemia, ET)和原發(fā)性骨髓纖維化(primary myelofibrosis, PMF)[68]。、、、等基因突變常見于骨髓增殖性腫瘤[69~71]，其中最高頻的突變是基因V617F的功能獲得突變[72]。

斑馬魚jak2a突變體可以模擬人JAK2功能獲得突變，表現(xiàn)為與人類PV高度相似的紅細(xì)胞擴(kuò)增[70]。在斑馬魚胚胎表達(dá)突變型人基因會(huì)導(dǎo)致胚胎出現(xiàn)類似于ET患者的血小板生成增加表型[73]。然而，以上兩種模型均是在斑馬魚胚胎瞬時(shí)過(guò)表達(dá)突變基因，并不能穩(wěn)定遺傳。

在內(nèi)皮細(xì)胞誘導(dǎo)突變基因表達(dá)，模擬MPN表型，表現(xiàn)為CHT區(qū)域紅系、髓系祖細(xì)胞顯著擴(kuò)增，腎髓中血細(xì)胞分化受阻，以及外周血中紅系、髓系祖細(xì)胞聚積[74]。斑馬魚突變體呈現(xiàn)MPN類似表型，具體表現(xiàn)為髓系前體細(xì)胞擴(kuò)增[75]。通過(guò)正向遺傳篩選獲得的魚系，表現(xiàn)為紅系、髓系細(xì)胞擴(kuò)增，且該表型依賴Flt3信號(hào)途徑[76]。這些模型模擬了人類MPN表型并初步闡釋了其發(fā)病機(jī)制。

2.2.2 骨髓增生異常綜合征模型

骨髓增生異常綜合征(myelodysplastic syndro-mes, MDS)是異質(zhì)性造血干細(xì)胞疾病，表現(xiàn)為病態(tài)造血或無(wú)效造血，以及高風(fēng)險(xiǎn)向AML轉(zhuǎn)化[77]。近年來(lái)大量基因組分析顯示，MDS某些亞型與剪接體(spliceosome)或表觀遺傳因子的突變密切相關(guān)[78~81]。

剪接體突變是MDS發(fā)病的關(guān)鍵因素，約60%的MDS患者中會(huì)發(fā)生不同形式的剪接體突變[79]。剪接因子3B亞單位1 (splicing factor 3B subunit 1, SF3B1)是MDS中最易突變的剪接體因子之一[79,81]。斑馬魚功能缺失突變體的初級(jí)造血受損，髓系和紅系細(xì)胞分化與增殖障礙，造血干祖細(xì)胞產(chǎn)生減少[82]，該表型與MDS患者癥狀類似[82]。正向遺傳篩選得到的()突變體具有HSC產(chǎn)生正常、而髓系和紅系細(xì)胞發(fā)育缺陷的表型，圖位克隆分析鑒定出剪接因子是其突變基因[83]。上述剪接因子突變體具有類似表型，但也有各自獨(dú)特的表現(xiàn)，與臨床觀察一致，即不同剪接因子突變的患者有共同的疾病特點(diǎn)，也有個(gè)體化的特征，斑馬魚模型的使用將有助于個(gè)體化精準(zhǔn)醫(yī)療的實(shí)施。

TET2功能缺失突變常見于髓系惡性腫瘤患者(約30%的MDS以及約10%的再發(fā)AML病例)[84]。Gjini等[85]研究表明，純合突變體的胚胎期造血正常，但隨著年齡的增長(zhǎng)會(huì)出現(xiàn)進(jìn)行性克隆性骨髓增生異常、貧血和髓系祖細(xì)胞擴(kuò)增；受精后24個(gè)月，它們呈現(xiàn)出更嚴(yán)重的MDS表型，如外周血紅細(xì)胞異常增生。

原癌基因是造血細(xì)胞增殖和分化的重要調(diào)控因子，其異常表達(dá)通常與多種血液疾病相關(guān)[86~88]。轉(zhuǎn)基因斑馬魚Tg (GFP)中基因過(guò)度表達(dá)，髓系細(xì)胞顯著擴(kuò)增，類似于人類MDS表型，且部分成魚會(huì)發(fā)展為AML和ALL。這是由于該模型中過(guò)度表達(dá)的基因影響細(xì)胞周期相關(guān)基因的表達(dá)，導(dǎo)致造血前體細(xì)胞過(guò)度增殖。靶向藥物flavopiridol可緩解c-myb胚胎和成魚的MDS樣癥狀。該模型可用于MDS發(fā)生分子機(jī)制的探究以及治療藥物的篩選[89]。

2.3 斑馬魚血液疾病模型的應(yīng)用

2.3.1 移植評(píng)價(jià)實(shí)驗(yàn)

應(yīng)用斑馬魚進(jìn)行血液腫瘤細(xì)胞移植可以用來(lái)定義和量化白血病增殖細(xì)胞(leukemia-propagating cells, LPCs)以及探究其啟動(dòng)白血病的潛能[90]。斑馬魚的移植評(píng)價(jià)實(shí)驗(yàn)具有諸多優(yōu)勢(shì)：使用受精后4周內(nèi)的斑馬魚胚胎進(jìn)行移植不需要免疫抑制，因?yàn)樵摃r(shí)期斑馬魚缺乏成熟的適應(yīng)性免疫系統(tǒng)[91]；透明的胚胎或成體魚與多種熒光轉(zhuǎn)基因品系相結(jié)合，促進(jìn)了活體成像技術(shù)在腫瘤發(fā)生發(fā)展研究中的應(yīng)用[92]。

首次移植實(shí)驗(yàn)是應(yīng)用T-ALL模型Tg ()，將EGFP標(biāo)記的白血病細(xì)胞移植到輻射后野生型成年斑馬魚腹腔內(nèi)，這些細(xì)胞在腹腔注射后14天開始擴(kuò)散，14~26天歸巢于胸腺[38]。連續(xù)移植后誘發(fā)疾病的能力是腫瘤細(xì)胞自我更新的標(biāo)志。T-ALL細(xì)胞連續(xù)移植揭示了大部分T-ALL細(xì)胞具有啟動(dòng)白血病的潛能。此外，大規(guī)模的單細(xì)胞移植實(shí)驗(yàn)則證實(shí)了這一啟動(dòng)潛能呈現(xiàn)差異性，即0.1%~ 15.9%甚至更少的白血病細(xì)胞有能力重建腫瘤[90]。

復(fù)發(fā)T-ALL患者中經(jīng)常發(fā)生克隆進(jìn)化，導(dǎo)致腫瘤更具侵襲性，這一現(xiàn)象與LPCs的遺傳多樣性及其增強(qiáng)的白血病啟動(dòng)潛能相關(guān)[93,94]。研究表明，Notch信號(hào)能促進(jìn)T-ALL癌前T細(xì)胞克隆擴(kuò)增，提高積累必要突變并完全轉(zhuǎn)化為L(zhǎng)PCs的可能性和速度[95]。大規(guī)模細(xì)胞移植篩選實(shí)驗(yàn)顯示單個(gè)克隆之間存在功能變異，少數(shù)克隆隨著時(shí)間的推移提高了生長(zhǎng)速度和擴(kuò)增潛力。Blackburn等[96]研究證明，克隆進(jìn)化激活A(yù)kt信號(hào)通路促進(jìn)T-ALL的生長(zhǎng)，同時(shí)可促使腫瘤細(xì)胞對(duì)地塞米松產(chǎn)生耐藥性。以上研究提示，克隆進(jìn)化促使T-ALL對(duì)化療產(chǎn)生耐藥性，而且這可能發(fā)生于藥物暴露之前。

斑馬魚移植受體的免疫系統(tǒng)可被地塞米松或γ射線短暫抑制，卻無(wú)法進(jìn)行長(zhǎng)期移植；且僅可用于同源移植，限制了移植模型的廣泛應(yīng)用[97]。為了解決這些問(wèn)題，美國(guó)哈佛大學(xué)David Langenau團(tuán)隊(duì)構(gòu)建了免疫缺陷斑馬魚模型—rag2突變體。該突變體中功能性T細(xì)胞和B細(xì)胞數(shù)量減少，但仍能存活和繁殖，并且可進(jìn)行多種組織與癌細(xì)胞長(zhǎng)期穩(wěn)定移植。但是該模型純合子斑馬魚不能繁殖，以及個(gè)體間B細(xì)胞缺陷差異極大，可能影響植入潛能[98]。免疫缺陷斑馬魚模型jak3突變體和prkdc突變體，分別導(dǎo)致T細(xì)胞與NK細(xì)胞、成熟T細(xì)胞和B細(xì)胞的缺失，兩種突變體均具有植入能力，但只有純合突變體可以繁殖，并且在細(xì)胞移植后存活[97]。斑馬魚prkdc，il2rg雙突變體中T細(xì)胞、B細(xì)胞和NK細(xì)胞缺陷，David Langenau團(tuán)隊(duì)?wèi)?yīng)用該免疫缺陷突變體建立并評(píng)估了腫瘤移植模型，能夠重現(xiàn)多種病人來(lái)源的腫瘤生長(zhǎng)遷移等情況，并可在單細(xì)胞水平進(jìn)行活體實(shí)時(shí)研究[99]。

2.3.2 化學(xué)藥物篩選

斑馬魚模型是進(jìn)行高通量藥物篩選的理想選擇，主要基于兩個(gè)優(yōu)點(diǎn)：(1)整體動(dòng)物模型，可以針對(duì)特定生物學(xué)事件發(fā)現(xiàn)活性化合物和藥物靶標(biāo)；(2)全面評(píng)估化合物的活性和副作用，排除具有明顯毒副作用的化合物，縮短藥物研發(fā)周期[100~102]。

研究表明前列腺素E2 (prostaglandin E2, PGE2)的代謝活性衍生物二甲基前列腺素E2 (16,16- dimethyl-PGE2, dmPGE2)可以增加HSCs數(shù)量，同時(shí)也可促進(jìn)輻射損傷成體魚中腎髓的恢復(fù)[103]。此外，dmPGE2可提升小鼠骨髓HSCs移植重建能力[103]。臨床前分析顯示，dmPGE2能顯著促進(jìn)體外人造血細(xì)胞集落形成，提高異種移植后人臍血干細(xì)胞(human cord blood, hCB)的植入效率[104]。人類臍帶血移植的臨床研究顯示，經(jīng)dmPGE2處理的臍帶血細(xì)胞具有持久、多系重建潛能，且安全性高，移植后患者中性粒細(xì)胞的恢復(fù)速度也大大加快[105]。美國(guó)猶他大學(xué)Nikolaus Trede團(tuán)隊(duì)?wèi)?yīng)用淋系轉(zhuǎn)基因斑馬魚進(jìn)行藥物篩選，確定小分子化合物L(fēng)enaldekar (LDK)可以有效消除不成熟的T細(xì)胞而不影響正常細(xì)胞的細(xì)胞周期，且可延長(zhǎng)大部分T-ALL成魚的生存期[106]。在小鼠模型中，LDK也表現(xiàn)出了減緩疾病進(jìn)展的功效。同時(shí)對(duì)于直接取自臨床原發(fā)性白血病(包括難治性B-ALL和慢性粒細(xì)胞白血病)患者的樣本，LDK可以殺死其中大部分的白血病細(xì)胞[106]。這項(xiàng)工作證明了使用斑馬魚篩選抗腫瘤藥物的實(shí)用性，同時(shí)LDK的發(fā)現(xiàn)也為白血病靶向治療提供了新方向[106]。

接受異種移植的斑馬魚也是藥物篩選的一個(gè)重要模型。將人類白血病細(xì)胞移植到斑馬魚胚胎中用于篩選非致畸的白血病治療藥物，結(jié)果顯示伊馬替尼和奧沙福林可消除白血病細(xì)胞，且對(duì)受體胚胎無(wú)毒性；而全反式維甲酸和4EGI-1表現(xiàn)出致畸作用，不能作為抗白血病藥物[107]。

3 結(jié)語(yǔ)與展望

斑馬魚血液疾病模型的應(yīng)用為人們了解疾病的病理生理學(xué)、基因型與表型的相關(guān)性以及探索治療方案提供了新的途徑。應(yīng)用正反向遺傳學(xué)方法獲得的斑馬魚血液疾病模型，可用于化學(xué)篩選靶向治療藥物，或進(jìn)行異體移植研究白血病增殖細(xì)胞的功能等(圖1)。然而斑馬魚模型的應(yīng)用通常缺乏對(duì)腫瘤發(fā)病的時(shí)空控制，這限制了其在腫瘤進(jìn)展和轉(zhuǎn)移研究中的應(yīng)用。最近一項(xiàng)研究開發(fā)了一種成年斑馬魚轉(zhuǎn)基因電穿孔(transgene electroporation in adult zebrafish, TEAZ)技術(shù)，可以在成魚體內(nèi)定時(shí)定點(diǎn)導(dǎo)入特定的DNA，如原癌基因、CRISPR/Cas9基因編輯組件以及特定啟動(dòng)子驅(qū)動(dòng)熒光蛋白的DNA載體[108]，可以實(shí)現(xiàn)在特定組織和發(fā)育時(shí)期引入遺傳改變。斑馬魚模型應(yīng)用的另一個(gè)限制是缺乏可靠的細(xì)胞表面標(biāo)記及抗體，目前大多數(shù)造血細(xì)胞分選都是基于轉(zhuǎn)基因標(biāo)記，極大地限制了特定譜系亞群的分選[109]。此外，人們對(duì)斑馬魚造血微環(huán)境的了解仍然很少，造血細(xì)胞之間的相互作用、與微環(huán)境細(xì)胞的相互作用及其與血液疾病的關(guān)系值得進(jìn)一步探究。

異種移植模型極大地加深了人們對(duì)白血病發(fā)生和干細(xì)胞生物學(xué)的理解?；颊邅?lái)源的異種移植物 保持了人類癌癥固有的克隆異質(zhì)性，提供了優(yōu)于體外系統(tǒng)的微環(huán)境，這對(duì)藥物的開發(fā)與臨床轉(zhuǎn)化有重要意義[110,111]。雖然斑馬魚異種移植平臺(tái)已初步建成[99,112,113]，但是人類造血干祖細(xì)胞在斑馬魚體內(nèi)僅能短暫地存活[114]。最近，一種人源化斑馬魚模型可以表達(dá)多種人類造血特異性的細(xì)胞因子，從而促進(jìn)了受體中造血干祖細(xì)胞的存活、自我更新和多向分化，且移植白血病細(xì)胞表現(xiàn)出向造血組織的歸巢，更準(zhǔn)確地模擬了人類白血病的行為[115]。除了血液系統(tǒng)腫瘤，在其他人源腫瘤的移植方面斑馬魚也顯示出獨(dú)特的優(yōu)勢(shì)：活體成像可以對(duì)腫瘤生長(zhǎng)、轉(zhuǎn)移、血管生成以及腫瘤起始細(xì)胞進(jìn)行動(dòng)態(tài)分析，斑馬魚也為高通量篩選抗癌藥物提供了一個(gè)經(jīng)濟(jì)有效的平臺(tái)[99,116,117]。

圖1 斑馬魚血液疾病模型構(gòu)建與應(yīng)用

應(yīng)用正反向遺傳學(xué)方法構(gòu)建斑馬魚血液疾病模型，可用于化學(xué)藥物篩選或腫瘤細(xì)胞移植評(píng)價(jià)研究。

疾病模型的應(yīng)用有助于探索個(gè)體化精準(zhǔn)醫(yī)療。最近，研究人員利用斑馬魚拯救了一位患有淋巴管疾病的12歲男孩的生命。全外顯子測(cè)序發(fā)現(xiàn)該患兒X染色體上基因發(fā)生錯(cuò)義突變，過(guò)表達(dá)攜帶這種突變的人源基因?qū)е掳唏R魚淋巴管的過(guò)度生長(zhǎng)，化學(xué)篩選證明MEK抑制劑對(duì)該表型有回救作用；這不僅明確了基因突變導(dǎo)致該患兒發(fā)生疾病，也尋找到了有效的治療藥物[118]。這是通過(guò)斑馬魚實(shí)現(xiàn)精準(zhǔn)醫(yī)療的成功案例，但是由于生命活動(dòng)的關(guān)聯(lián)性與復(fù)雜性，基因的改變往往會(huì)造成級(jí)聯(lián)反應(yīng)，如何將斑馬魚的研究成果過(guò)渡到人類醫(yī)療中仍然需要持續(xù)的探索。

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Zebrafish blood disease models and applications

Jiani Guo, Fan Liu, Lu Wang

,,,,,

Hematopoiesis is a complex, orderly and conserved developmental process, coordinated by multiple factors including transcription factors and signaling pathways. Dysregulation of any of these factors may cause developmental or functional defects in the blood system, leading to the pathogenesis of blood diseases. Zebrafish hematopoiesis and the underlying molecular mechanisms are highly conserved with those in mammals. The use of zebrafish to recapitulate abnormal changes in pathogenic factors can build models of related blood diseases, thus providing powerful tools for exploring the molecular mechanisms of pathogenesis and progression, visualization of tumorigenesis and high-throughput chemical screening. In this review, we summarize the zebrafish models of blood diseases and their applications. These disease models not only help to improve our understanding of the pathophysiology of the blood system and the molecular mechanisms on pathogeneses of blood diseases, but also provide new ideas for the treatment of clinically relevant hematological malignancies.

zebrafish; blood diseases; animal model; chemical screens

2020-05-25;

2020-06-27

國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(編號(hào)：2018YFA0801200)，國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：31771604)和中國(guó)醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程項(xiàng)目(編號(hào)：2019-I2M-1-006)資助[Supported by the Ministry of Science and Technology of China (No. 2018YFA0801200), the National Natural Science Foundation of China (No. 31771604), and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (No. 2019-I2M-1-006)]

郭佳妮，在讀碩士研究生，專業(yè)方向：干細(xì)胞與再生醫(yī)學(xué)。E-mail: gjn2205150205@126.com

王璐，博士，研究員，研究方向：干細(xì)胞與再生醫(yī)學(xué)。E-mail: wanglu1@ihcams.ac.cn

10.16288/j.yczz.20-148

2020/8/10 13:51:05

URI: https://kns.cnki.net/kcms/detail/11.1913.R.20200806.1704.001.html

(責(zé)任編委: 劉峰)