王寶菊， 朱 彬， 郭偉娜， 楊東亮

(華中科技大學同濟醫(yī)學院附屬協(xié)和醫(yī)院 感染病科， 武漢 430022)

HBV感染與復制模型的建立及應用

王寶菊， 朱 彬， 郭偉娜， 楊東亮

(華中科技大學同濟醫(yī)學院附屬協(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復制和致病機制，探索新的治療靶點，進而研發(fā)新的治療藥物或方案。合適的HBV感染與復制模型是上述研究的基礎。由于HBV具有嚴格的種屬限制性及組織親嗜性，使得HBV感染與復制模型的研發(fā)受到一定限制。在國家傳染病科技重大專項資助下，國內(nèi)研究者建立了一系列的細胞和動物模型，就此并結(jié)合近年來國內(nèi)外研究進展進行簡要綜述。

肝炎病毒， 乙型； 細胞模型； 疾病模型， 動物

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。我國是乙型肝炎大國，雖然乙型肝炎疫苗的計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域亟待解決的問題。由于HBV感染具有嚴格的種屬限制性和組織親嗜性，HBV感染與復制模型研究受到很大限制，尤其我國在該領域的研究相對滯后。在國家傳染病科技重大專項課題的資助下，國內(nèi)學者近年來在HBV感染與復制模型的建立及應用方面取得了較大進展，本文就此并結(jié)合國內(nèi)外近年來進展簡要總結(jié)如下。

1 細胞模型

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

表1 HBV感染與復制細胞模型的比較

1.1 HBV感染的細胞模型

1.1.1 人/樹鼩原代肝細胞 HBV能夠感染高度分化的原代人肝細胞(primary human hepatocytes, PHH)。然而，PHH很難獲得，且在體外培養(yǎng)條件下，會逐漸失去對HBV的易感性。原代樹鼩肝細胞(primary tupaia hepatocytes, PTH)可作為體外研究HBV感染的替代模型?；赑TH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PHH的培養(yǎng)條件可以延長PHH HBV感染的時間窗，如胡康洪等通過三維培養(yǎng)技術將PHH對HBV易感的時間窗延長至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)新了胚胎肝細胞的凍存方法，并發(fā)現(xiàn)凍存的胚胎肝細胞與非實質(zhì)性肝細胞同時培養(yǎng)可使胚胎肝細胞迅速成熟且對HBV的易感性持續(xù)至10周[3]。

1.1.2 HepaRG細胞和HLCZ01 HepaRG細胞是人肝臟前體細胞，來源于丙型肝炎肝癌患者的肝組織，保留了許多原代肝細胞的特征，包括關鍵代謝酶、藥物轉(zhuǎn)運蛋白以及核受體的表達[4]。HepaRG細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細胞(dHepaRG)可持續(xù)產(chǎn)生感染性HBV顆粒100 d以上[5]。HepaRG細胞HBV感染系統(tǒng)已成為一種公認的可用于抗病毒藥物研發(fā)以及評估的有效工具[6]。朱海珍等[7]近年建立了一株來源于HCV相關肝癌樣本的肝癌細胞系(HLCZ01)，不僅可以被HBV感染，也對HCV易感，且無需分化誘導處理便可支持長達90 d的病毒感染。

1.1.3 表達NTCP的肝癌細胞 李文輝等發(fā)現(xiàn)NTCP是HBV進入的功能性受體?；谶@一發(fā)現(xiàn)，有學者[8]構(gòu)建了可組成性表達NTCP的肝癌細胞，并證實大部分轉(zhuǎn)染了NTCP的肝癌細胞對HBV易感。應用NTCP穩(wěn)定轉(zhuǎn)染的HepG2細胞，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，最近的實驗顯示環(huán)孢素[10]及其衍生物[11]可以與NTCP直接作用并干擾HBV進入易感肝細胞。

有趣的是，表達NTCP的小鼠肝細胞可以支持HDV感染，但是卻不能支持HBV感染[12]。進一步研究[13]顯示，小鼠肝細胞對HBV感染的限制性也許發(fā)生在進入之后、病毒轉(zhuǎn)錄之前，敲除抗病毒信號通路中的幾大類已知的分子對于這種限制性并無影響。

1.2 HBV復制細胞模型 通過將含有超長HBV基因組的重組質(zhì)粒穩(wěn)定轉(zhuǎn)染至人肝癌細胞系所獲得的能夠支持HBV穩(wěn)定復制的肝癌細胞系，如眾所周知的HepG2.2.15和HepAD38細胞系，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)定復制細胞系，上述細胞系均已申報專利并完成中國典型培養(yǎng)物保藏中心生物典藏。應用上述細胞系的研究發(fā)現(xiàn)CRISPR-Cas9可以清除細胞系中整合的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 動物模型

由于HBV感染具有嚴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物模型。將HBV基因?qū)肷臣毎旧w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所建立的復制模型僅能重現(xiàn)部分HBV生命周期，對HBV并不易感。盡管如此，上述動物模型已被廣泛應用于HBV研究的各個領域(表2)。

2.1 HBV感染的動物模型

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，同時伴隨肝臟炎癥及與HBV感染患者相似的免疫應答，是目前最理想的模擬HBV自然感染的動物模型[20]。已被用于研究HBV感染發(fā)病機制、評價抗病毒藥物和治療性疫苗效果[21-22]。但是，由于動物保護及費用等原因，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，該動物是否能夠作為HBV感染模型仍有待進一步研究。

2.1.2 樹鼩模型 樹鼩(Tupaiabelangeri)，屬樹鼩科樹鼩屬，主要分布在我國西南省份和東南亞各國。其可作為HAV和HBV感染模型，在HBV感染的基礎上加上黃曲霉毒素誘導的肝癌模型也被用于研究肝癌發(fā)病機制[4-26]。由于樹鼩是野生動物，個體差異較大，成年動物人工感染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，我國學者建立了較大規(guī)模人工繁育樹鼩種群，深入開展了遺傳學和基因組學的系統(tǒng)研究，采用近親繁殖和基因工程改造技術，有可能發(fā)展為可以廣泛應用的實驗室動物，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感染與復制動物模型的比較

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)移植人肝細胞可使FRG小鼠中人肝細胞達到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，實現(xiàn)了規(guī)?；a(chǎn)，并用于評價靶向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胞，因此不適用于研究免疫應答及免疫治療策略。研究者通過多種策略構(gòu)建了人免疫細胞和人肝細胞雙嵌合小鼠模型，即人源化小鼠模型，如AFC8和A2/NSG小鼠模型。已被用來進行HCV感染、HBV感染以及肝臟炎癥及纖維化機制研究[37-38]。

2.2 嗜肝DNA病毒感染的動物模型

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價抗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)病機制研究方面，應用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池并不會因HBV DNA復制水平而改變[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)病機制、評價抗病毒藥物以及免疫預防和治療策略[44-47]。筆者所在研究組長期從事土撥鼠模型研究，發(fā)現(xiàn)我國喜馬拉雅旱獺(Marmotahimalayana)與土撥鼠是同屬動物，對WHV高度易感[48]。已建立中國喜馬拉雅旱獺養(yǎng)殖基地及種群，并開展了實驗動物化的相關研究。比較了土撥鼠與旱獺肝脾組織的轉(zhuǎn)錄組學數(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90%以上；對20余種土撥鼠和旱獺免疫相關分子等進行了克隆、同源性分析、表達以及抗體制備；完善了WHV感染血清和組織病毒學指標檢測技術以及免疫應答分析技術，為旱獺WHV感染模型的標準化奠定了基礎；同時該模型也被用于抗病毒藥物研發(fā)以及新的預防和治療策略的研究，例如對基于核苷類藥物的HBV暴露后預防替代策略進行評價，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單用的部分動物及與DNA疫苗聯(lián)用的所有動物均產(chǎn)生保護性免疫[49-62]。

2.3 HBV復制的動物模型

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價GM-CSF和HBV S基因融合的DNA疫苗[63]；田志剛教授亦應用該模型進行了肝細胞和血清蛋白組學[64]、自然殺傷細胞參與CCL4加速HBV轉(zhuǎn)基因小鼠肝纖維化進程的相關研究[65]。WHV轉(zhuǎn)基因小鼠也已用于抗HBV治療新技術研究[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復制模型，改變注射劑量可以影響復制持續(xù)時間[67]。通過尾靜脈在短時間內(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)運至肝細胞內(nèi)。Chisari首次通過高壓水注射方法將含有復制性HBV基因組的質(zhì)粒轉(zhuǎn)運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的表達水平及復制持續(xù)時間。陳培哲等[68]采用pAAV/HBV1.2質(zhì)粒和C57BL/6小鼠可使HBV復制長達6個月，而采用pAAV/HBV1.2質(zhì)粒和C3H/HeN小鼠則可使HBV復制時間延長至46周。筆者課題組對該模型進行了深入研究，完善了制備技術并將其用于乙型肝炎發(fā)病機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淋巴細胞雖然缺乏活化和增殖表型，但仍能抑制效應性T淋巴細胞應答；Poly(I∶C)通過IFN依賴的途徑促進HBV清除；IFNα對不同基因型HBV的抗病毒效應存在差異；不同免疫抑制劑對HBV復制及肝內(nèi)HBV特異性免疫應答的影響不同等[69-73]。

上海巴斯德研究所鄧強研究團隊[14]將一段含有重組位點的外源內(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表達的情況下，在肝細胞中可誘導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胞中迅速積累，并誘導完整、有效的HBV復制。研究表明小鼠體內(nèi)的T淋巴細胞免疫反應被特異性激活但并不完全。進一步減少DNA注射劑量，小鼠體內(nèi)HBV復制則顯著延長并伴隨肝臟持續(xù)性炎癥損傷。

3 小結(jié)

總之，近年來國內(nèi)外在HBV復制/感染細胞和動物模型研究方面均取得了顯著進展，并有力地促進了HBV相關研究。但是，現(xiàn)有的HBV復制/感染模型均存在一定的局限性，如何進一步優(yōu)化和完善已有的HBV復制/感染模型，推進標準化和規(guī)?；?，為我國病毒性肝炎基礎研究、疫苗和新藥研發(fā)等提供全方位的模型平臺支持，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感染與復制模型的建立及應用[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。

國家傳染病科技重大專項(2008ZX10002011，2012ZX10004503)；國家自然科學基金(81001313，81101248，81371828，81461130019)；國家國際科技合作計劃(2011DFA31030)；國家科技支撐計劃課題(2015BAI09B06)；德國科學基金會德中跨學科重大合作項目(TRR60)

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

楊東亮，電子信箱：dlyang@hust.edu.cn。

R512.62

A

1001-5256(2017)08-1458-07