用rAAV8-1.3HBV制备两种品系小鼠乙型肝炎病毒感染模型的比较研究  

Study on the Differences of Two Mouse Models of Hepatitis B Virus Infection by Transduction with rAAV8-1.3HBV

作　　者：王刚[1] 董小岩 田文洪[1,3] 尉迟捷[1] 郑刚 高杰 王国婧[1] 魏国超[1] 周育森[4] 吴小兵[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]北京五加和分子医学研究所,北京100176 [3]吉林大学生命科学学院,长春130012 [4]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《病毒学报》2012年第5期541-547,共7页Chinese Journal of Virology

基　　金：肝炎相关肝癌生物治疗新途径新制品的研究和应用(No.2008ZX10002-023);新药创制科技重大专项(No.2009ZX09102-237)

摘　　要：我们先前用rAAV8-1.3HBV静脉注射C57BL/6小鼠成功地制备了慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染模型。为了探讨不同品系的小鼠对rAAV8-1.3HBV静脉注射是否具有不同反应,本研究比较了C57BL/6和BALB/c小鼠静脉注射重组病毒后外周血中HBV抗原和抗体水平、病毒载量和肝脏组织HBcAg表达情况,以及不同剂量重组病毒注射与这些指标的关系。将低(4×109 Viral genome,vg)、中(4×1010vg)和高(4×1011vg)三种剂量的rAAV8-1.3HBV通过尾静脉注射至C57BL/6和BALB/c小鼠,分别利用ELISA和荧光定量PCR方法检测血清中的HBV抗原、抗体水平以及HBV DNA,利用免疫组化检测肝脏组织HBcAg的表达。结果发现,对于C57BL/6小鼠,三种不同剂量rAAV8-1.3HBV注射均可造成100%小鼠出现HBV持续感染;血清HBsAg、HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,其表达水平随重组病毒注射剂量的增加而升高,高剂量注射时可造成超过40%的肝细胞感染HBV,血清中HBV DNA可达105 IU/mL以上;未检测到针对HBV的抗体。对于BALB/c小鼠,三种不同剂量rAAV8-1.3HBV注射也可造成100%小鼠出现HBV持续感染;血清HBeAg和HBV DNA以及肝组织HBcAg稳定表达超过8个月,但是血清HBsAg在重组病毒注射2周之后显著下降甚至消失;在中剂量注射组的BALB/c小鼠血清中检测到低水平的Anti-HBs;血清HBeAg和肝组织HBcAg的表达水平随重组病毒注射剂量的增加而增高,并且各剂量组表达水平均高于C57BL/6小鼠,高剂量注射时可造成超过50%的肝细胞感染HBV。本研究表明,低至4×109 vg剂量的rAAV8-1.3HBV注射即可造成C57BL/6和BALB/c两种品系小鼠出现HBV持续感染,并且HBV复制水平随重组病毒注射剂量增加而增高;BALB/c小鼠对HBV的免疫反应强于C57BL/6小鼠,可以产生针对HBsAg的体液免疫反应而使血清HBsAg转阴,但无法清除携带HBV的肝细胞。We recently developed a mouse model of hepatitis B virus （HBV） chronic infection＇by intrave- nous（i, v. ） injection with rAAV8-1.3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1.3HBV ,we intravenously injected rAAV8-1.3HBV at doses of 4× 10^9 （Viral genome,vg）,4×10^11 vg and 4×10^11vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8- 1.3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1.3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1.3HBV at the dose of 4×10^11 vg,over 40 % of hepatocytes expressed HBcAg and serum viral DNA reached over 10^5 IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1.3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-pos- itive hepatocytes in BALB/c mice increased in a rAAV8-1.3HBV dose-dependent manner. Injection with rAAVS-1.3HBV at the dose of 4 × 10^11 vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAVS-1.3HBV injection at the dose of 4 ×10^10 vg. In conclusion,both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by L v. injection with rAAV8-1.3HBV at doses of 4 × 10^9-4 × 10^11 vg and the levels of HBV replication increase in a rAAVS-1.3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg,but fail to mediate HBV clearance.

关 键 词：动物模型 乙型肝炎病毒 重组8型腺相关病毒 小鼠品系 

分 类 号：Q78[生物学—分子生物学]