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

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。

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

We recently developed a mouse model of hepatitis B virus(HBV) chronic infection by intravenous(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 4×109(Viral genome,vg),4×1010vg and 4×1011vg 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 4×1011vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 105IU/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-positive hepatocytes in BALB/c mice increased in a rAAV8-1.3HBV dose-dependent manner.Injection with rAAV8-1.3HBV at the dose of 4×1011vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg.Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1.3HBV injection at the dose of 4×1010vg.In conclusion,both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i.v.injection with rAAV8-1.3HBV at doses of 4×109~4×1011vg and the levels of HBV replication increase in a rAAV8-1.3HBV 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.