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黄恺,孙鑫,赵志敏,彭渊,陶艳艳,刘成海.两种乙肝肝纤维化小鼠复合模型的比较[J].中国实验动物学报,2019,27(5):598~603.
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
投稿时间:2019-03-20  
DOI:10. 3969 / j.issn.1005-4847. 2019. 05. 008
中文关键词:  动物模型  乙型肝炎病毒  炎症  肝纤维化  病理学
英文关键词:mouse model  hepatitis B virus  inflammation  hepatic fibrosis  histopathology
基金项目:
作者单位E-mail
黄恺 上海市中医临床重点实验室,上海 201203 kanghui12@ 163.com 
孙鑫 上海中医药大学附属曙光医院肝病研究所,上海 201203  
赵志敏 上海市中医临床重点实验室,上海 201203  
彭渊 上海中医药大学附属曙光医院肝病研究所,上海 201203  
陶艳艳 上海中医药大学附属曙光医院肝病研究所,上海 201203  
刘成海 1. 上海市中医临床重点实验室,上海 201203
2. 上海中医药大学附属曙光医院肝病研究所,上海 201203 
chenghailiu@ hotmail.com 
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中文摘要:
      目的 采用C57BL/6 N-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/6 N-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?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. 0×104 IU/ 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?
英文摘要:
      Objective We examined the virological, biochemical and pathological features of two compositemouse models of hepatitis B fibrosis. Methods Mice were transfected with rAAV8-1. 3HBV adeno-associated virus andC57BL/6 N-Tg(1. 28HBV) / Vst type hepatitis B virus transgenic mice, combined with CCl4 to induce hepatitis B liverfibrosis in mice. The mice were divided into the following groups: wild-type control group ( WT), rAAV8-1. 3HBVtransfection control group (rAAV), CCl4 control group (CCl4 ), rAAV8-1. 3HBV transfection with CCl4 model group(rAAV+CCl4), C57BL/6 N-Tg (1. 28HBV) / Vst hepatitis B virus transgenic control group (Tg), and HBV transgenic complex CCl4 model group (Tg+CCl4 ).After 12 weeks,the levels of HBsAg and HBeAg were measured by ELISA. TheHBV-DNA load was detected by PCR. The expression of HBsAg and HBcAg in the liver tissue was observed byimmunohistochemical 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 toobserve the pathological changes. Results ELISA result showed positive serum HBsAg and HBeAg,meanwhile the PCRexamined the HBV-DNA load was higher than 1. 0×104 IU/ mL in all the groups except for the WT and CCl4 group. TheHBsAg and HBeAg content in the Tg+CCl4 group was higher than in the rAAV+CCl4 group. The levels of HBV-DNA in therAAV and rAAV+CCl4 groups were higher than those in the Tg and Tg+CCl4 groups. Immunohistochemistry showed positiveHBsAg and HBcAg in the liver tissues in all groups except the WT group and CCl4 group. The expression of HBsAg wasdecreased but HBcAg was significantly increased in the rAAV+group compared with the Tg group. No significant differencewas detected in HBsAg between the Tg+CCl4 group and rAAV+CCl4 group, but the HBcAg positive expression wasobviously increased in the latter group. The levels of ALT, AST and liver Hyp were significantly increased in the CCl4group, Tg+CCl4 group and rAAV+CCl4 group. The Hyp content in the Tg+CCl4 group was higher than in the rAAV+CCl4group. The biopsy showed that there were significantly increased inflammation reaction and collagen deposition in the CCl4group, Tg+CCl4 group and rAAV+CCl4 group. The Tg+CCl4 group collagen deposition was higher than in the rAAV+CCl4group. Conclusions The two composite mouse models conform to the hepatits B liver fibrosis model and the differences areconcentrated in virological indicators and pathological changes. The rAAV + CCl4 group has an advantage in HBV-DNA load, and the Tg + CCl4 group was more obvious in fibrosis progress.
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