CN113637706A - 乙型肝炎病毒重组cccDNA小鼠模型的建立方法及用途 - Google Patents
乙型肝炎病毒重组cccDNA小鼠模型的建立方法及用途 Download PDFInfo
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Abstract
本发明属微生物动物细胞系技术领域，具体涉及一种8型腺相关病毒载体介导建立的乙型肝炎病毒重组cccDNA(rcccDNA)小鼠模型及其建立方法和用途。本发明利用8型腺相关病毒载体，将两端带有loxP位点的HBV单拷贝基因组带入Alb‑Cre转基因小鼠体内，建立了AAV‑rcccDNA小鼠模型。建立的AAV‑rcccDNA小鼠模型具有1)建模成功率高；2)模型小鼠肝内rcccDNA表达水平高、维持时间长，易于用Southern blot方法进行检测；3)模型小鼠血清及肝内病毒抗原表达水平、维持时间长；4)模型小鼠肝内HBV特异性T细胞免疫反应偏弱等特点。本发明建立的小鼠模型适用于筛选和测试HBV cccDNA靶向药物、免疫调节剂以及研究慢乙肝治愈的合适模型中。
Description
技术领域
本发明属于微生物动物细胞系技术领域，涉及乙型肝炎病毒重组cccDNA(rcccDNA)小鼠模型，具体涉及一种8型腺相关病毒载体介导建立的乙型肝炎病毒重组cccDNA(rcccDNA)小鼠模型及其建立方法和用途。
背景技术
现有技术公开了乙型肝炎病毒(Hepatitis B virus，HBV)感染引起的乙型肝炎是严重危害人类健康的重大传染病。据统计，慢性乙型肝炎(Chronic hepatitis B，CHB)感染患者罹患肝硬化、肝衰竭和肝癌的风险较正常人显著增高(>20％)。目前，我国有超过的9000万的慢乙肝患者，其中2800万需要治疗，虽然近年来慢乙肝的治疗取得了一定的进展，但治疗疗程、耐药及停药后复发等一系列问题仍是目前困扰临床的难题，其中一个重要的原因即是HBV特征性的结构分子“共价闭合环状DNA(covalently closed circularDNA，cccDNA)”的持续存在和难以清除。
研究显示，HBV cccDNA由病毒基因组松弛环状DNA(relaxed circular DNA，rcDNA)在肝细胞核内经过一系列步骤形成，其中rcDNA可来源于新感染的病毒颗粒或病毒复制过程中新合成的病毒核心颗粒；HBV cccDNA转录生成病毒mRNA，其中前基因组RNA(pregenomic RNA，pgRNA)是病毒蛋白翻译和逆转录复制的模板，因此，只有清除细胞核内的cccDNA，才有实现完全治愈慢性乙型肝炎的可能，这也是HBV新药研发追求的目标。
目前本领域对cccDNA的形成、功能活性和稳定性调控等生物学特性的认识仍存在许多空白。研究实践显示，HBV动物模型的发展受制于HBV严格的种特异性，小鼠作为使用量最大、研究最详尽的哺乳类实验动物，不易感染HBV；即便是在人牛磺胆酸钠联合转运多肽(hNTCP)转基因小鼠模型中，由于cccDNA形成效率低下，仍然无法建立起有效感染。目前大多数HBV小鼠模型依赖于转基因、病毒载体转导或高压尾静脉注射的方法建立，在这些模型中很难检测到cccDNA。人肝嵌合鼠是唯一有效支持HBV感染和cccDNA形成的小鼠模型，但模型建立过程中高度的技术挑战以及模型鼠适应性免疫的缺乏均为限制此类模型普遍性应用的重要因素。因此，建立一种易于培育、具有较稳定的cccDNA库及正常免疫活性的小鼠模型，是乙肝研究领域的迫切需求。
近年来，重组cccDNA(rcccDNA)小鼠模型的到了较快的发展，rcccDNA能够有效支持病毒的复制和蛋白表达，建立慢性感染，然而在已报道的rcccDNA小鼠模型中，鼠肝细胞内rcccDNA库的水平和持续时间仍然不适用于cccDNA靶向研究，虽然在建模一周内Southern blot可以清楚地检测到rcccDNA条带，但之后rcccDNA水平迅速下降，限制了它们在研究cccDNA相关的生物学机制和开发新的cccDNA靶向药物中的应用。
基于腺相关病毒(AAV)具有感染率高、无致癌隐患、免疫原性低等特点，其中，8型对肝脏的亲和力最高，本发明在Cre/loxP系统体内生成rcccDNA的策略基础上，利用8型腺相关病毒载体(AAV8)导入的方式，建立了一个新型的HBV rcccDNA小鼠模型。该模型有望成为筛选和测试cccDNA靶向药物和研究慢乙肝治愈的合适模型。
发明内容
本发明的目的在于克服现有技术中HBV rcccDNA小鼠模型的不足，建立一种具有较高表达水平和较长维持时间的rcccDNA的小鼠模型，该小鼠模型能够用于HBV cccDNA相关生物学特性研究和抗HBV药物，特别是直接靶向于cccDNA的新型药物的筛选评价。
具体的，本发明提供了乙型肝炎病毒重组cccDNA(rcccDNA)小鼠模型及其建立方法，利用腺相关病毒(AAV)载体构建乙型肝炎病毒(HBV)重组共价、闭合、环状DNA(recombinant covalently closed circular DNA,rcccDNA)的小鼠模型。
更具体的，本发明提供了一种8型腺相关病毒载体介导建立的乙型肝炎病毒重组cccDNA(rcccDNA)小鼠模型及其建立方法和用途。
本发明中，采用8型腺相关病毒载体，将两端带有loxP位点的HBV单拷贝基因组带入Alb-Cre转基因小鼠体内，建立了AAV-rcccDNA小鼠模型。
本发明中，对建立的AAV-rcccDNA小鼠模型肝内rcccDNA的表达水平和维持时间进行了检测；结果显示，8周龄雄性Alb-Cre小鼠接受2×1011vg重组病毒尾静脉注射建模后，其肝内的rcccDNA在30周内，易于通过Southern blot方法进行检测。
本发明中，对所述的AAV-rcccDNA小鼠血清和肝内病毒学指标进行了检测，结果显示，所述的模型小鼠血清HBsAg、HBeAg，肝内HBsAg、HBcAg的表达均可维持一年以上。
本发明中，对所述的AAV-rcccDNA小鼠模型的肝内免疫学和病理学进行了检测，结果显示，所述的模型鼠肝内HBV特异性T细胞免疫反应偏弱，也无明显的炎性反应，但可观测到轻微的纤维化增生及代谢水平变化。
本发明进一步提供了所建立的AAV-rcccDNA小鼠模型在用于HBVcccDNA相关生物学特性研究和抗HBV药物，特别是直接靶向于cccDNA的新型药物的筛选评价中的用途。本发明的实施例中，在AAV8-rcccDNA模型鼠中评价了AKSZ0002-B、Bay41-4109、恩替卡韦(ETV)的抗病毒效果，结果显示，所述的模型小鼠血清和肝内病毒指标的变化能很好地反映出这三种抗病毒制剂的不同作用机制。
本发明建立了一个新型的HBV rcccDNA小鼠模型，该模型将成为筛选和测试cccDNA靶向药物和研究慢乙肝治愈的合适模型。
附图说明
图1.AAV-rcccDNA小鼠模型肝内rcccDNA生成的模式图。
图2.AAV-rcccDNA小鼠模型肝内rcccDNA生成检测。
图3.AAV-rcccDNA小鼠模型血清和肝内病毒指标检测。
图4.AAV-rcccDNA小鼠模型肝内HBV特异性T淋巴细胞表达和功能检测。
图5.AAV-rcccDNA小鼠模型用于抗病毒效果评价。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围，本发明的保护范围也不限于下述的实施例。
实施例1AAV-rcccDNA小鼠模型的建立
①pAAV8-rcccDNA质粒构建：AAV8载体由pAAV8-CAG-RFP质粒经XhoI/BamHI双酶切获得，两端带有loxP位点的HBV单拷贝基因组序列由prcccDNA质粒扩增而来，以重组法克隆入pAAV8载体；
②病毒包装：pAAV8-rcccDNA、pRC和pHelper质粒共转HEK293T细胞，转染试剂选用PEI。转染72小时后，将细胞连同培养基一同收集到50ml离心管中；
③病毒纯化：采用氯仿处理-PEG/NaCl沉淀-氯仿抽提法进行纯化，收集细胞及培养液，加入1/10体积的氯仿，置于37℃摇床中剧烈振摇1h，加入固体氯化钠至终浓度1mol/L，振摇溶解，4℃，12000r/min离心15min，取出上层水相，加PEG8000至终浓度10％(w/v)，振摇融解，冰浴放置1h，11000r/min离心15min，弃上清，用适量PBS将离心管底和管壁上的沉淀吹打洗脱下来分装至1.5ml离心管中，此时的rAAV定义为中间产物，再经benzonase核酸酶(50U/ml)，室温消化30min，加入等体积的氯仿抽提，即获得浓缩和纯化的重组腺相关病毒液，采用定量PCR法进行定量，以pAA V8-rcccDNA质粒梯度稀释后作为标准曲线；
④模型建立：将AAV8-rcccDNA病毒按注射剂量采用尾静脉注射Alb-Cre雄性小鼠，病毒以PBS稀释至200μl/只进行注射，Alb-Cre转基因小鼠肝脏特异性地表达重组酶Cre，因此在小鼠肝细胞内，Cre酶对loxP位点进行识别和切割，单拷贝的HBV基因组将从病毒载体上释放下来，单独环化形成rcccDNA(如图1所示)。
实施例2 AAV8-rcccDNA小鼠模型肝内rcccDNA检测
①模型建立：8周龄的雄性Alb-Cre小鼠接受2×1011vg AAV8-rcccDNA病毒注射后，于相应时间点处死分离肝脏；
②抽提DNA：称取100mg的小鼠肝组织，置于冰预冷的100μl PBS中，用剪刀尽可能地剪成小块，用组织研磨器进行研磨，过100μm孔径细胞筛网，获得肝细胞悬液。采用Hirt抽提法抽提；
③Southern blot检测rcccDNA：采用Roche公司的地高辛探针标记和检测系统进行Southern Blot检测(DIG High Prime DNA Labeling and Detection Starter Kit IImanual)；在30周内，可检测到rcccDNA的明显信号；rcccDNA条带可被EcoRI内切酶线性化，但对T5核酸外切酶不敏感的特性，证实了其超螺旋的结构(如图2A所示)；
④定量PCR检测rcccDNA：采用针对于rcccDNA上特异性内含子序列的引物进行检测，在建模后第52周仍能通过定量PCR检测到rcccDNA的特异性信号(如图2B所示)，通过对定量数据的拟合分析，AAV8-rcccDNA小鼠肝内rcccDNA的半衰期为7.8周(6.113-10.64周)。
实施例3 AAV8-rcccDNA小鼠模型血清及肝内病毒学指标检测
①模型建立：指定周龄的雄性Alb-Cre小鼠接受指定剂量的AAV8-rcccDNA病毒注射建立模型；
②血清病毒指标检测：注射后每两周进行一次眼眶下眦静脉丛采血，采血量为200μl不抗凝全血，采集到的血液在室温放置2小时后，8000rpm室温离心10分钟分离血清，用于HBsAg、HBeAg、谷丙转氨酶ALT、HBV DNA检测，结果显示，AAV-rcccDNA小鼠模型的血清HBsAg、HBeAg分别在104IU/ml和103COI水平(如图3A，B所示)，并在整个实验期间(51周)内保持阳性；血清HBV DNA起始量在104IU/ml，而后逐渐下降(如图3C所示)，AAV8-rcccDNA小鼠模型血清病毒抗原和DNA的表达水平与接受注射的重组病毒剂量相关，而与小鼠的周龄关系不大；
③肝内病毒指标检测：在相应时间点处死小鼠，采用心脏灌流后，分离肝、肾等器官/组织，各器官/组织取一部分置于4％多聚甲醛固定液中，进行石蜡包埋、切片，用于免疫组化染色等，结果显示，AAV-rcccDNA小鼠肝内HBsAg和HBcAg也呈长期表达(如图3D所示)，HBsAg信号定位于胞浆，而HBcAg信号呈多态性，胞浆强染、胞核强染与核浆均染色较强的细胞均可观察到，建模早期(2周)HBsAg和HBcAg的阳性表达面积分别为60.71±6.73％和17.43±6.27％，晚期(51周)分别为22.83±4.24％和12.04±1.07％。
实施例4 AAV8-rcccDNA小鼠模型肝内HBV特异性T淋巴细胞表达和功能检测
①模型建立：8周龄的雄性Alb-Cre小鼠接受1×1012vg重组病毒注射建模；另设三组对照：PBS组；EmptyAAV组：尾静脉接受1×1012vg的空载AAV8病毒注射；pAAV-rcccDNA组，高压尾静脉接受10μg的pAAV-rcccDNA质粒注射；
②乙肝特异性CD8+T淋巴细胞数量检测：建模二周时处死小鼠，分离肝淋巴细胞，以CD3、CD8、HBsAg或HBcAg特异性Tetramer进行表面染色，流式检测乙肝特异性CD8+T淋巴细胞的比例，结果显示，AAV8-rcccDNA模型小鼠肝内HBsAg和HBcAg特异性CD8+ T淋巴细胞的比例都要显著低于质粒组(Env353 4.62±1.52％vs20.77±4.98％，p＝0.0047；Core930.919±0.34％vs 3.91±0.59％,p＝0.0017)。而PBS组及AAV空载病毒组小鼠体内都没有明显的HBsAg或HBcAg特异性CD8+T淋巴细胞的表达(如图4A所示)；
③乙肝特异性CD8+T淋巴细胞功能检测：将分离得到的各组建模二周时的小鼠肝淋巴细胞，用HBsAg肽段刺激5小时后，内因子染色检测IFN-γ和TNF-α阳性T淋巴细胞的比例，结果显示，AAV8-rcccDNA模型小鼠肝淋巴细胞经S肽段刺激后，IFN-γ和TNF-α阳性T淋巴细胞的比例要显著低于质粒组(IFN-γ2.27±0.47％vs 5.78±0.99％，p＝0.023；TNF-α0.48±0.06％vs 1.94±0.56％，p＝0.004)(如图4B所示)；进一步检测PD-1分子在AAV8-rcccDNA模型小鼠肝内Env353特异性CD8T+细胞上的表达水平，结果显示AAV-rcccDNA组PD-1的表达水平(2309.33±77.56)显著高于质粒组(1377±189)，表明AAV-rcccDNA转基因小鼠肝内HBV特异性CD8T+细胞功能受损(如图4C所示)。
实施例5 AAV-rcccDNA小鼠模型用于抗病毒效果评价
①模型建立：8周龄雄性Alb-Cre小鼠经2×1011vg AAV8-rcccDNA病毒注射建模后四周，将小鼠随机分为对照组、HBV表达抑制剂AKSZ0002-B处理组、核衣壳组装抑制剂Bay41-4109处理组及逆转录抑制剂ETV处理组，AKSZ0002-B和Bay41-4109属于两类新型制剂，二者分别在降低HBsAg表达和抑制病毒复制等指标上显示出良好的效果，但对肝内cccDNA的影响却鲜有检测，AKSZ0002-B组给予4mg/kg/只/次的药物灌胃，一天两次，Bay41-4109组给予15mg/kg/只/次的药物灌胃，一天两次，ETV组给予0.03mg/kg/只/次的药物灌胃，一天一次，对照组给予含30％PEG400、0.2％DMSO的PBS灌胃，200μl/只/次，一天两次，所有受试化合物以指定的剂量和频率给药28天，第28天，每组处死部分小鼠，其余小鼠停药观测至49天；
②样本收取及检测：每周采血监测血清抗原和病毒载量，第28天，每组处死部分小鼠，取肝脏，部分肝脏用于固定、石蜡包埋、切片及HBsAg、Core免疫组化染色，其余保存于RNAlater中，用于Hirt抽提及Southern blot检测，结果显示，AKEX0007组给药一周后，小鼠血清中HBsAg显著降低(降低1.6log10，p＝0.002)，此后维持在较低水平(如图5A，左所示)；HBeAg水平降低0.35log10(p<0.001)(如图5A，中间所示)；血清HBV DNA略微降低了0.1log10(如图5A，右所示)，相比之下，ETV组仅观察到血清HBV-DNA显著下降(降低1.17log10，p＝0.007)，而血清HBsAg、HBeAg无明显变化，Bay41-4109组的血清病毒指标变化与ETV组相似(如图5B所示)，血清病毒抗原水平无显著变化，但病毒载量下降(降低0.88log10，p＝0.005)；免疫组化结果显示，与对照组小鼠相比，AKEX0007组小鼠肝内HBsAg显著下降，HBcAg略有下降(HBsAg 39.32±3.5％vs13.55±5.43％，HBcAg 13.32±2.69％vs 9.7±1.82％)(如图5C所示)；Bay41-4109组肝内HBcAg显著下降(17.68±2％vs 8.89±1.13％)(如图5D所示)；而ETV组两种病毒抗原均无明显变化(如图5C所示)；进一步对各组小鼠肝脏rcccDNA水平的监测显示，与对照组相比，在治疗结束时(如图5E-F所示)和随访结束时(如图5G所示)，各组小鼠肝内的rcccDNA水平均无显著变化；与此结果一致，在停止给药后AKEX0007和ETV组可观察到HBV血清标志物明显反弹至基线水平(如图5A所示)。
综上，AAV8-rcccDNA小鼠模型在血清和肝内病毒学指标上能够很好地反映受试的三种抗病毒制剂的不同作用机制。
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但显然本领域的技术人员可根据本文说明，在本发明的范围内对本发明做出一些修改的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (6)
1.一种乙型肝炎病毒重组cccDNA小鼠模型的建立方法，其特征在于，利用8型腺相关病毒载体，将两端带有loxP位点的HBV单拷贝基因组带入Alb-Cre转基因小鼠体内，利用8型腺相关病毒载体介导构建乙型肝炎病毒HBV重组共价、闭合、环状DNA(rcccDNA)小鼠模型，通过下述步骤：
①pAAV8-rcccDNA质粒构建；
②病毒包装：pAAV8-rcccDNA、pRC和pHelper质粒共转HEK293T细胞；
③病毒纯化：采用氯仿处理-PEG/NaCl沉淀-氯仿抽提法进行纯化，获得浓缩和纯化的重组腺相关病毒液，定量PCR法定量，以pAAV8-rcccDNA质粒梯度稀释后作为标准曲线；
④模型建立：将AAV8-rcccDNA病毒按注射剂量采用尾静脉注射Alb-Cre雄性小鼠，单拷贝的HBV基因组将从病毒载体上释放下来，单独环化形成rcccDNA。
2.按权利要求1所述的建立方法，其特征在于，所建立的小鼠模型AAV-rcccDNA，其生物学特性为：
1)建模后得模型鼠肝内rcccDNA表达水平高、维持时间长，在30周内易于通过Southernblot方法进行检测；
2)模型鼠血清及肝内病毒学指标表达水平高、维持时间长；
3)模型鼠肝内HBV特异性T细胞免疫反应偏弱，无明显的炎性反应，能观测到轻微的纤维化增生及代谢水平变化。
3.按权利要求1所述的建立方法，其特征在于，所建立的小鼠模型AAV-rcccDNA在用于制备HBV cccDNA相关生物学研究模型中的用途。
4.按权利要求1所述的建立方法，其特征在于，所建立的小鼠模型AAV-rcccDNA在用于制备HBV cccDNA相关免疫学特性研究模型中的用途。
5.按权利要求1所述的建立方法，其特征在于，所建立的小鼠模型AAV-rcccDNA在用于制备筛选抗HBV药物模型中的用途。
6.按权利要求1所述的建立方法，其特征在于，所建立的小鼠模型AAV-rcccDNA在用于制备评价抗HBV药物物模型中的用途。
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