两种乙肝肝纤维化小鼠复合模型的比较

Comparison of two composite mouse models of hepatitis B fibrosis

目的采用C57BL/6N-Tg(1.28HBV)/Vst型乙肝病毒转基因小鼠与rAAV8-1.3HBV腺相关病毒转染小鼠联合CCl4腹腔注射诱导乙肝肝纤维化小鼠模型,比较两种小鼠模型的病毒学及生化病理特点。方法实验分野生型对照组(WT)、rAAV8-1.3HBV转染组(rAAV)、CCl4组(CCl4)、rAAV8-1.3HBV复合CCl4模型组(rAAV+CCl4),C57BL/6N-Tg(1.28HBV)/Vst乙肝病毒转基因组(Tg)、转基因复合CCl4组(Tg+CCl4),共计造模12周。ELISA试剂盒测定血清HBsAg、HBeAg水平,荧光定量PCR法检测血清HBV-DNA载量,肝组织石蜡切片免疫组化染色观察肝内HBsAg及HBcAg的表达,生化试剂盒测定血清ALT、AST、AKP,盐酸水解法检测肝组织羟脯氨酸(Hyp)含量。HE染色及天狼星红染色观察炎症与胶原等病理变化。[JP2]结果除WT组、CCl4组,其余各组ELISA检测血清HBsAg、HBeAg均呈阳性,同时荧光定量PCR检测HBV-DNA载量均高于1.0×104IU/mL。其中Tg+CCl4组HBsAg、HBeAg含量高于rAAV+CCl4组。rAAV、rAAV+CCl4组HBV-DNA水平高于Tg、Tg+CCl4组。免疫组化表明:除WT组、CCl4组,其余各组肝组织中HBsAg与HBcAg均呈阳性。rAAV组对比Tg组,HBsAg表达减少;rAAV组相比Tg组,HBcAg表达明显增多。Tg+CCl4与rAAV+CCl4相比,HBsAg结果无明显差异,rAAV+CCl4组HBcAg阳性表达明显增高。生化结果显示:CCl4组、Tg+CCl4组、rAAV+CCl4组血清ALT、AST及肝组织Hyp含量均显著升高;其中Tg+CCl4组Hyp含量高于rAAV+CCl4组。AKP结果对比WT组无明显差异。病理结果表明:对比WT组,rAAV与Tg组炎症与胶原沉积不明显;CCl4组、Tg+CCl4组、rAAV+CCl4组炎症反应及胶原沉积明显增高,其中Tg+CCl4组胶原沉积高于rAAV+CCl4组。结论两种复合模型符合乙肝肝纤维化模型需求,其差异主要集中于病毒学指标以及病理改变。rAAV+CCl4组在HBV-DNA载量上具有优势,Tg+CCl4组在纤维化进展更为明显。

Objective We examined the virological,biochemical and pathological features of two composite mouse models of hepatitis B fibrosis.Methods Mice were transfected with rAAV8-1.3HBV adeno-associated virus and C57BL/6 N-Tg (1.28HBV)/Vst type hepatitis B virus transgenic mice,combined with CCl 4 to induce hepatitis B liver fibrosis in mice.The mice were divided into the following groups: wild-type control group (WT),rAAV8-1.3HBV transfection control group (rAAV), CCl 4 control group (CCl 4),rAAV8-1.3HBV transfection with CCl 4 model group (rAAV+CCl 4),C57BL/6 N-Tg (1.28HBV)/Vst hepatitis B virus transgenic control group (Tg),and HBV transgenic complex CCl 4 model group (Tg+CCl 4).After12 weeks,the levels of HBsAg and HBeAg were measured by ELISA. The HBV-DNA load was detected by PCR.The expression of HBsAg and HBcAg in the liver tissue was observed by immunohistochemical staining.Serum ALT,AST and AKP were determined by a biochemical kit,and hydroxyproline (Hyp) content was detected by hydrochloric acid hydrolysis method. HE staining and Sirius red staining were used to observe the pathological changes.Results ELISA result showed positive serum HBsAg and HBeAg,meanwhile the PCR examined the HBV-DNA load was higher than1.0×10 4 IU/mL in all the groups except for the WT and CCl 4 group.The HBsAg and HBeAg content in the Tg+CCl 4 group was higher than in the rAAV+CCl 4 group.The levels of HBV-DNA in the rAAV and rAAV+CCl 4 groups were higher than those in the Tg and Tg+CCl 4 groups. Immunohistochemistry showed positive HBsAg and HBcAg in the liver tissues in all groups except the WT group and CCl 4 group.The expression of HBsAg was decreased but HBcAg was significantly increased in the rAAV+group compared with the Tg group.No significant difference was detected in HBsAg between the Tg+CCl 4 group and rAAV+CCl 4 group,but the HBcAg positive expression was obviously increased in the latter group.The levels of ALT,AST and liver Hyp were significantly increased in the CCl 4 group,Tg+CCl 4 group and rAAV+CCl 4 group.The Hyp content in the Tg+CCl 4 group was higher than in the rAAV+CCl 4 group.The biopsy showed that there were significantly increased inflammation reaction and collagen deposition in the CCl 4 group,Tg+CCl 4 group and rAAV+CCl 4 group.The Tg+CCl 4 group collagen deposition was higher than in the rAAV+CCl 4 group.Conclusions The two composite mouse models conform to the hepatits B liver fibrosis model and the differences are concentrated in virological indicators and pathological changes.The rAAV + CCl 4 group has an advantage in HBV-DNA load,and the Tg + CCl 4 group was more obvious in fibrosis progress.