HBV感染與復(fù)制模型的建立及應(yīng)用

王寶菊， 朱 彬， 郭偉娜， 楊東亮

(華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬協(xié)和醫(yī)院 感染病科， 武漢 430022)

HBV感染與復(fù)制模型的建立及應(yīng)用

王寶菊， 朱 彬， 郭偉娜， 楊東亮

(華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬協(xié)和醫(yī)院 感染病科， 武漢 430022)

乙型肝炎是危害人類健康的重要傳染病，目前的抗病毒治療，如干擾素、核苷和核苷酸類藥物仍無法治愈慢性乙型肝炎。因此，亟待闡明HBV復(fù)制和致病機(jī)制，探索新的治療靶點(diǎn)，進(jìn)而研發(fā)新的治療藥物或方案。合適的HBV感染與復(fù)制模型是上述研究的基礎(chǔ)。由于HBV具有嚴(yán)格的種屬限制性及組織親嗜性，使得HBV感染與復(fù)制模型的研發(fā)受到一定限制。在國(guó)家傳染病科技重大專項(xiàng)資助下，國(guó)內(nèi)研究者建立了一系列的細(xì)胞和動(dòng)物模型，就此并結(jié)合近年來國(guó)內(nèi)外研究進(jìn)展進(jìn)行簡(jiǎn)要綜述。

肝炎病毒， 乙型； 細(xì)胞模型； 疾病模型， 動(dòng)物

全球有1/3的人口正在或曾經(jīng)感染HBV，其中3.5億為慢性感染者。我國(guó)是乙型肝炎大國(guó)，雖然乙型肝炎疫苗的計(jì)劃接種大大減少了青少年人群HBV感染率，但仍有約9300萬慢性HBV感染者[1]?，F(xiàn)有的抗HBV藥物如干擾素、核苷和核苷酸類藥物存在應(yīng)答率不高或不能有效清除HBV等問題。因此，為了使更多患者實(shí)現(xiàn)臨床治愈，探索新的藥物或治療方案是乙型肝炎研究領(lǐng)域亟待解決的問題。由于HBV感染具有嚴(yán)格的種屬限制性和組織親嗜性，HBV感染與復(fù)制模型研究受到很大限制，尤其我國(guó)在該領(lǐng)域的研究相對(duì)滯后。在國(guó)家傳染病科技重大專項(xiàng)課題的資助下，國(guó)內(nèi)學(xué)者近年來在HBV感染與復(fù)制模型的建立及應(yīng)用方面取得了較大進(jìn)展，本文就此并結(jié)合國(guó)內(nèi)外近年來進(jìn)展簡(jiǎn)要總結(jié)如下。

1 細(xì)胞模型

HBV感染與復(fù)制體外模型包括人/樹鼩原代肝細(xì)胞、來源于肝癌組織的HepaRG細(xì)胞和HLCZ01細(xì)胞、表達(dá)鈉離子-?；悄懰峁厕D(zhuǎn)運(yùn)蛋白(sodium taurocholate cotransporting polypeptide, NTCP)的肝癌細(xì)胞系、HepG2.2.15和HepAD38細(xì)胞系，以及HepG2.4D14、HepG2.A64、HepG2.C5細(xì)胞系等。這些HBV感染與復(fù)制細(xì)胞模型在闡明HBV與肝細(xì)胞膜表面受體及相關(guān)分子結(jié)構(gòu)相互作用，HBV被細(xì)胞膜內(nèi)吞并進(jìn)入細(xì)胞的過程、HBV cccDNA合成、病毒基因復(fù)制和轉(zhuǎn)錄，病毒包裝和病毒釋放機(jī)制，以及HBV致病機(jī)制等方面發(fā)揮了重要作用。目前已經(jīng)被廣泛使用。當(dāng)然，上述細(xì)胞模型也存在不同的問題與不足(表1)。

表1 HBV感染與復(fù)制細(xì)胞模型的比較

1.1 HBV感染的細(xì)胞模型

1.1.1 人/樹鼩原代肝細(xì)胞 HBV能夠感染高度分化的原代人肝細(xì)胞(primary human hepatocytes, PHH)。然而，PHH很難獲得，且在體外培養(yǎng)條件下，會(huì)逐漸失去對(duì)HBV的易感性。原代樹鼩肝細(xì)胞(primary tupaia hepatocytes, PTH)可作為體外研究HBV感染的替代模型。基于PTH模型的研究，李文輝等發(fā)現(xiàn)NTCP是HBV的受體。也有研究者發(fā)現(xiàn)，通過改進(jìn)PHH的培養(yǎng)條件可以延長(zhǎng)PHH HBV感染的時(shí)間窗，如胡康洪等通過三維培養(yǎng)技術(shù)將PHH對(duì)HBV易感的時(shí)間窗延長(zhǎng)至3周(未發(fā)表數(shù)據(jù))。為了克服PHH難以獲得的問題，胡康洪等[2]還創(chuàng)新了胚胎肝細(xì)胞的凍存方法，并發(fā)現(xiàn)凍存的胚胎肝細(xì)胞與非實(shí)質(zhì)性肝細(xì)胞同時(shí)培養(yǎng)可使胚胎肝細(xì)胞迅速成熟且對(duì)HBV的易感性持續(xù)至10周[3]。

1.1.2 HepaRG細(xì)胞和HLCZ01 HepaRG細(xì)胞是人肝臟前體細(xì)胞，來源于丙型肝炎肝癌患者的肝組織，保留了許多原代肝細(xì)胞的特征，包括關(guān)鍵代謝酶、藥物轉(zhuǎn)運(yùn)蛋白以及核受體的表達(dá)[4]。HepaRG細(xì)胞在特定條件下培養(yǎng)幾周后可以被HBV感染。盡管感染效率并不高，也未觀察到病毒播散，但分化的HepaRG細(xì)胞(dHepaRG)可持續(xù)產(chǎn)生感染性HBV顆粒100 d以上[5]。HepaRG細(xì)胞HBV感染系統(tǒng)已成為一種公認(rèn)的可用于抗病毒藥物研發(fā)以及評(píng)估的有效工具[6]。朱海珍等[7]近年建立了一株來源于HCV相關(guān)肝癌樣本的肝癌細(xì)胞系(HLCZ01)，不僅可以被HBV感染，也對(duì)HCV易感，且無需分化誘導(dǎo)處理便可支持長(zhǎng)達(dá)90 d的病毒感染。

1.1.3 表達(dá)NTCP的肝癌細(xì)胞 李文輝等發(fā)現(xiàn)NTCP是HBV進(jìn)入的功能性受體?；谶@一發(fā)現(xiàn)，有學(xué)者[8]構(gòu)建了可組成性表達(dá)NTCP的肝癌細(xì)胞，并證實(shí)大部分轉(zhuǎn)染了NTCP的肝癌細(xì)胞對(duì)HBV易感。應(yīng)用NTCP穩(wěn)定轉(zhuǎn)染的HepG2細(xì)胞，Ko等[9]發(fā)現(xiàn)DEAD盒RNA解旋酶家族成員DDX3是影響cccDNA轉(zhuǎn)錄的宿主限制性因素。另外，最近的實(shí)驗(yàn)顯示環(huán)孢素[10]及其衍生物[11]可以與NTCP直接作用并干擾HBV進(jìn)入易感肝細(xì)胞。

有趣的是，表達(dá)NTCP的小鼠肝細(xì)胞可以支持HDV感染，但是卻不能支持HBV感染[12]。進(jìn)一步研究[13]顯示，小鼠肝細(xì)胞對(duì)HBV感染的限制性也許發(fā)生在進(jìn)入之后、病毒轉(zhuǎn)錄之前，敲除抗病毒信號(hào)通路中的幾大類已知的分子對(duì)于這種限制性并無影響。

1.2 HBV復(fù)制細(xì)胞模型 通過將含有超長(zhǎng)HBV基因組的重組質(zhì)粒穩(wěn)定轉(zhuǎn)染至人肝癌細(xì)胞系所獲得的能夠支持HBV穩(wěn)定復(fù)制的肝癌細(xì)胞系，如眾所周知的HepG2.2.15和HepAD38細(xì)胞系，已被廣泛用于篩選抗HBV藥物[14]、制備HBV[15]、研究病毒宿主相互作用[16]。徐東平等建立了基于我國(guó)流行的C基因型野生株、阿德福韋酯(ADV)耐藥株、恩替卡韋(ETV)耐藥株、多重耐藥株等一系列HBV穩(wěn)定復(fù)制細(xì)胞系，上述細(xì)胞系均已申報(bào)專利并完成中國(guó)典型培養(yǎng)物保藏中心生物典藏。應(yīng)用上述細(xì)胞系的研究發(fā)現(xiàn)CRISPR-Cas9可以清除細(xì)胞系中整合的HBV DNA[17]、多藥耐藥蛋白4可能影響核苷類藥物的抗病毒效果[18]、HBV通過microRNA-15a-Smad7-TGFβ通路影響凋亡及腫瘤發(fā)生等[19]。

2 動(dòng)物模型

由于HBV感染具有嚴(yán)格的種屬限制性和組織親嗜性，目前僅黑猩猩、樹鼩和人源化小鼠肝臟可被HBV感染。鑒于嗜肝DNA病毒家族成員鴨乙型肝炎病毒(duck hepatitis B virus, DHBV)和土撥鼠肝炎病毒(woodchuck hepatitis virus, WHV) 與HBV高度同源，且能自然感染鴨和土撥鼠，因此，鴨和土撥鼠模型也屬于感染動(dòng)物模型。將HBV基因?qū)肷臣?xì)胞染色體建立的轉(zhuǎn)基因小鼠或者通過尾靜脈注射方式將HBV基因?qū)敫闻K所建立的復(fù)制模型僅能重現(xiàn)部分HBV生命周期，對(duì)HBV并不易感。盡管如此，上述動(dòng)物模型已被廣泛應(yīng)用于HBV研究的各個(gè)領(lǐng)域(表2)。

2.1 HBV感染的動(dòng)物模型

2.1.1 黑猩猩模型 黑猩猩接種HBV血清后可發(fā)展為急、慢性HBV感染，同時(shí)伴隨肝臟炎癥及與HBV感染患者相似的免疫應(yīng)答，是目前最理想的模擬HBV自然感染的動(dòng)物模型[20]。已被用于研究HBV感染發(fā)病機(jī)制、評(píng)價(jià)抗病毒藥物和治療性疫苗效果[21-22]。但是，由于動(dòng)物保護(hù)及費(fèi)用等原因，黑猩猩模型無法廣泛使用。Dupinay等[23]發(fā)現(xiàn)毛里求斯島的食蟹猴自然持續(xù)感染可能來自人類的HBV，該動(dòng)物是否能夠作為HBV感染模型仍有待進(jìn)一步研究。

2.1.2 樹鼩模型 樹鼩(Tupaiabelangeri)，屬樹鼩科樹鼩屬，主要分布在我國(guó)西南省份和東南亞各國(guó)。其可作為HAV和HBV感染模型，在HBV感染的基礎(chǔ)上加上黃曲霉毒素誘導(dǎo)的肝癌模型也被用于研究肝癌發(fā)病機(jī)制[4-26]。由于樹鼩是野生動(dòng)物，個(gè)體差異較大，成年動(dòng)物人工感染HBV后多表現(xiàn)為急性自限性感染，較少形成慢性化。但是幼齡期感染HBV則較容易形成慢性感染，與人類感染相似[27]。近年來，我國(guó)學(xué)者建立了較大規(guī)模人工繁育樹鼩種群，深入開展了遺傳學(xué)和基因組學(xué)的系統(tǒng)研究，采用近親繁殖和基因工程改造技術(shù)，有可能發(fā)展為可以廣泛應(yīng)用的實(shí)驗(yàn)室動(dòng)物，用于HBV感染、糖尿病、非酒精性脂肪肝等疾病的研究[28-34]。

表2 HBV感染與復(fù)制動(dòng)物模型的比較

2.1.3 人源化小鼠模型 最早的人肝嵌合小鼠模型使用的是尿激酶型纖溶酶原激活物(uPA)轉(zhuǎn)基因的免疫缺陷(Rag2-/-，SCID、SCID/beige)小鼠。人肝細(xì)胞移植至uPA-SCID小鼠后可獲得重建率較高的人源化肝臟模型，且能夠支持HBV和HCV感染。隨后在延胡索酰乙酰乙酸水解酶(Fah)缺陷的小鼠 (Fah-/-/Rag2-/-/IL2rg-/-，F(xiàn)RG)移植人肝細(xì)胞可使FRG小鼠中人肝細(xì)胞達(dá)到95%[35]。程通等[36]建立了基于FRG小鼠的HBV感染小鼠模型，優(yōu)化模型制備流程，實(shí)現(xiàn)了規(guī)?；a(chǎn)，并用于評(píng)價(jià)靶向HBsAg特殊表位的治療性抗體的療效。鑒于上述模型均缺乏人免疫細(xì)胞，因此不適用于研究免疫應(yīng)答及免疫治療策略。研究者通過多種策略構(gòu)建了人免疫細(xì)胞和人肝細(xì)胞雙嵌合小鼠模型，即人源化小鼠模型，如AFC8和A2/NSG小鼠模型。已被用來進(jìn)行HCV感染、HBV感染以及肝臟炎癥及纖維化機(jī)制研究[37-38]。

2.2 嗜肝DNA病毒感染的動(dòng)物模型

2.2.1 DHBV感染的鴨模型 DHBV為嗜肝DNA病毒中的禽類嗜肝DNA病毒，可自然感染部分種類鴨子。郝友華等[39]發(fā)現(xiàn)不同鴨種DHBV的自然感染率不同，通過腹腔和靜脈注射雛鴨DHBV感染率不同。DHBV持續(xù)感染的鴨模型被廣泛用于評(píng)價(jià)抗HBV藥物及聯(lián)合治療策略，如臨床常用的核苷類藥物ETV、核衣殼組裝抑制劑等以及抗病毒聯(lián)合免疫的治療策略[40]。在發(fā)病機(jī)制研究方面，應(yīng)用DHBV感染的鴨模型，發(fā)現(xiàn)早期天然免疫(非獲得性免疫缺失)是導(dǎo)致雛鴨DHBV持續(xù)感染的原因[41]，通過比較抗病毒治療及未經(jīng)治療的鴨肝內(nèi)cccDNA含量發(fā)現(xiàn)，cccDNA池并不會(huì)因HBV DNA復(fù)制水平而改變[42]，持續(xù)的DNA疫苗聯(lián)合IL-2和IFNγ質(zhì)粒注射能夠顯著減少鴨肝內(nèi)cccDNA，但仍無法實(shí)現(xiàn)徹底清除[43]。

2.2.2 WHV感染的土撥鼠模型 WHV是嗜肝DNA病毒中的正嗜肝DNA病毒。WHV不僅在病毒學(xué)特征方面與HBV高度近似，且其感染后的自然史與人感染HBV高度近似，因此被廣泛用于研究HBV發(fā)病機(jī)制、評(píng)價(jià)抗病毒藥物以及免疫預(yù)防和治療策略[44-47]。筆者所在研究組長(zhǎng)期從事土撥鼠模型研究，發(fā)現(xiàn)我國(guó)喜馬拉雅旱獺(Marmotahimalayana)與土撥鼠是同屬動(dòng)物，對(duì)WHV高度易感[48]。已建立中國(guó)喜馬拉雅旱獺養(yǎng)殖基地及種群，并開展了實(shí)驗(yàn)動(dòng)物化的相關(guān)研究。比較了土撥鼠與旱獺肝脾組織的轉(zhuǎn)錄組學(xué)數(shù)據(jù)，發(fā)現(xiàn)土撥鼠和旱獺超過75%的不同功能分子的同源性高達(dá)90%以上；對(duì)20余種土撥鼠和旱獺免疫相關(guān)分子等進(jìn)行了克隆、同源性分析、表達(dá)以及抗體制備；完善了WHV感染血清和組織病毒學(xué)指標(biāo)檢測(cè)技術(shù)以及免疫應(yīng)答分析技術(shù)，為旱獺WHV感染模型的標(biāo)準(zhǔn)化奠定了基礎(chǔ)；同時(shí)該模型也被用于抗病毒藥物研發(fā)以及新的預(yù)防和治療策略的研究，例如對(duì)基于核苷類藥物的HBV暴露后預(yù)防替代策略進(jìn)行評(píng)價(jià)，結(jié)果發(fā)現(xiàn)ETV單用或與核心蛋白DNA疫苗聯(lián)用均能完全阻斷WHV感染，并且ETV單用的部分動(dòng)物及與DNA疫苗聯(lián)用的所有動(dòng)物均產(chǎn)生保護(hù)性免疫[49-62]。

2.3 HBV復(fù)制的動(dòng)物模型

2.3.1 HBV轉(zhuǎn)基因小鼠模型 HBV轉(zhuǎn)基因小鼠已被廣泛用于HBV發(fā)病機(jī)制和抗病毒藥物研究?？紫槠降冉⒘?種特殊免疫遺傳背景的HBV轉(zhuǎn)基因小鼠——C57BL/6背景的HBV轉(zhuǎn)基因小鼠、HLA-A2/HBV和Rag1-/-/HBV雙轉(zhuǎn)基因小鼠，并廣泛提供其他研究者使用。任紅教授將該模型用于評(píng)價(jià)GM-CSF和HBV S基因融合的DNA疫苗[63]；田志剛教授亦應(yīng)用該模型進(jìn)行了肝細(xì)胞和血清蛋白組學(xué)[64]、自然殺傷細(xì)胞參與CCL4加速HBV轉(zhuǎn)基因小鼠肝纖維化進(jìn)程的相關(guān)研究[65]。WHV轉(zhuǎn)基因小鼠也已用于抗HBV治療新技術(shù)研究[66]。

2.3.2 HBV轉(zhuǎn)染小鼠模型 靜脈注射含HBV基因組的腺病毒載體(Ad-HBV)，可以在小鼠體內(nèi)建立HBV復(fù)制模型，改變注射劑量可以影響復(fù)制持續(xù)時(shí)間[67]。通過尾靜脈在短時(shí)間內(nèi)將大量含有裸DNA的液體注入小鼠體內(nèi)的方法稱為高壓水注射，能夠有效地將外源基因轉(zhuǎn)運(yùn)至肝細(xì)胞內(nèi)。Chisari首次通過高壓水注射方法將含有復(fù)制性HBV基因組的質(zhì)粒轉(zhuǎn)運(yùn)至免疫功能正常的小鼠肝內(nèi)，病毒血癥可持續(xù)約1周。此后研究表明，質(zhì)粒骨架、小鼠品系、性別以及注射劑量均可影響小鼠體內(nèi)HBV的表達(dá)水平及復(fù)制持續(xù)時(shí)間。陳培哲等[68]采用pAAV/HBV1.2質(zhì)粒和C57BL/6小鼠可使HBV復(fù)制長(zhǎng)達(dá)6個(gè)月，而采用pAAV/HBV1.2質(zhì)粒和C3H/HeN小鼠則可使HBV復(fù)制時(shí)間延長(zhǎng)至46周。筆者課題組對(duì)該模型進(jìn)行了深入研究，完善了制備技術(shù)并將其用于乙型肝炎發(fā)病機(jī)制和抗病毒治療策略等研究，如發(fā)現(xiàn)了肝內(nèi)的自然調(diào)節(jié)性T淋巴細(xì)胞雖然缺乏活化和增殖表型，但仍能抑制效應(yīng)性T淋巴細(xì)胞應(yīng)答；Poly(I∶C)通過IFN依賴的途徑促進(jìn)HBV清除；IFNα對(duì)不同基因型HBV的抗病毒效應(yīng)存在差異；不同免疫抑制劑對(duì)HBV復(fù)制及肝內(nèi)HBV特異性免疫應(yīng)答的影響不同等[69-73]。

上海巴斯德研究所鄧強(qiáng)研究團(tuán)隊(duì)[14]將一段含有重組位點(diǎn)的外源內(nèi)含子序列插入單拷貝的HBV基因組中，構(gòu)建出cccDNA前體質(zhì)粒(prcccDNA)。prcccDNA在重組酶Cre表達(dá)的情況下，在肝細(xì)胞中可誘導(dǎo)HBV重組cccDNA(rcccDNA)并以微染色體的形式存在。通過尾靜脈高壓水注射方式，rcccDNA可在免疫健全小鼠的肝細(xì)胞中迅速積累，并誘導(dǎo)完整、有效的HBV復(fù)制。研究表明小鼠體內(nèi)的T淋巴細(xì)胞免疫反應(yīng)被特異性激活但并不完全。進(jìn)一步減少DNA注射劑量，小鼠體內(nèi)HBV復(fù)制則顯著延長(zhǎng)并伴隨肝臟持續(xù)性炎癥損傷。

3 小結(jié)

總之，近年來國(guó)內(nèi)外在HBV復(fù)制/感染細(xì)胞和動(dòng)物模型研究方面均取得了顯著進(jìn)展，并有力地促進(jìn)了HBV相關(guān)研究。但是，現(xiàn)有的HBV復(fù)制/感染模型均存在一定的局限性，如何進(jìn)一步優(yōu)化和完善已有的HBV復(fù)制/感染模型，推進(jìn)標(biāo)準(zhǔn)化和規(guī)模化，為我國(guó)病毒性肝炎基礎(chǔ)研究、疫苗和新藥研發(fā)等提供全方位的模型平臺(tái)支持，是未來值得深入研究的重要方向。

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引證本文：WANG BJ, ZHU B, GUO WN, et al. Establishment and application of in vitro and in vivo models of hepatitis B virus infection[J]. J Clin Hepatol, 2017, 33(8): 1458-1464. (in Chinese) 王寶菊， 朱彬， 郭偉娜， 等. HBV感染與復(fù)制模型的建立及應(yīng)用[J]. 臨床肝膽病雜志, 2017, 33(8): 1458-1464.

(本文編輯：葛 俊)

Establishment and application of in vitro and in vivo models of hepatitis B virus infection

WANGBaoju,ZHUBin,GUOWeina,etal.

(DepartmentofInfectiousDiseases,UnionHospitalAffiliatedtoTongjiMedicalCollegeofHuazhongUniversityofScienceandTechnology,Wuhan430022,China)

Hepatitis B is still an important infectious disease which threatens human health, and current antiviral therapy, including interferon and nucleos(t)ide analogues, cannot cure chronic hepatitis B. Therefore, it is urgent to explore the detail mechanisms of HBV replication and pathogenesis, identify new therapeutic targets, and develop new drugs or treatment regimens, which relies on the development of suitable models for HBV infection and replication. Species restriction and tissue tropism of HBV have limited the development of models for HBV infection and replication. With the support by National Science and Technology Major Project for Infectious Diseases, the researchers in China have developed a series of cellular and animal models for HBV. This article reviews these models with reference to recent research advances in China and foreign countries.

hepatitis B virus; cell model; disease models, animal

10.3969/j.issn.1001-5256.2017.08.009

2017-06-30；

2017-07-12。

國(guó)家傳染病科技重大專項(xiàng)(2008ZX10002011，2012ZX10004503)；國(guó)家自然科學(xué)基金(81001313，81101248，81371828，81461130019)；國(guó)家國(guó)際科技合作計(jì)劃(2011DFA31030)；國(guó)家科技支撐計(jì)劃課題(2015BAI09B06)；德國(guó)科學(xué)基金會(huì)德中跨學(xué)科重大合作項(xiàng)目(TRR60)

王寶菊(1973-)，女，副教授，博士，主要從事乙型肝炎動(dòng)物模型及發(fā)病機(jī)制的研究。

楊東亮，電子信箱：dlyang@hust.edu.cn。

R512.62

A

1001-5256(2017)08-1458-07