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利用乙型肝炎病毒共价闭合环状DNA构建慢性乙型肝炎病毒感染的小鼠模型
Establishment of Hepatitis B Virus Chronic Infection Mouse Model Induced by HBV cccDNA
严磊1, 赵婷婷1, 王会娟1, 李小松2, 黄爱龙2, 谭毅1, 赖国旗1*
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DOI:
作者单位:1. 重庆医科大学实验动物中心, 重庆 400016;
2. 重庆医科大学感染性疾病分子生物学教育部重点实验室, 重庆 400016
中文关键字:乙型肝炎病毒;HBV共价闭合环状DNA;水动力法;小鼠模型
英文关键字:hepatitis B virus; HBV cccDNA; hydrodynamic; mouse model
中文摘要:目的 通过水动力法注射乙型肝炎病毒(HBV) 共价闭合环状DNA(cccDNA)构建C57BL/6小鼠慢性乙型肝炎病毒感染的模型。方法 取29只C57BL/6小鼠,分为实验组、对照组和空白组,应用水动力法分别注射HBV cccDNA、pAAV-HBV1.2及等渗盐水,于注射后收集不同时间点的血清和肝组织。利用放射免疫法检测血清样本中乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒e抗原(HBeAg);荧光定量PCR检测血清和肝组织中HBV DNA拷贝数;免疫组织化学法检测肝组织中HBsAg和乙型肝炎病毒核心抗原(HBcAg)的表达;苏木精-伊红(HE)染色观察肝组织病理变化;使用SPSS 17.0对数据进行统计学分析。结果 实验组HBsAg和HBeAg表达均呈现4个上升-下降曲线:HBsAg峰值分别出现在第3天、第3周、第7周和第9周;HBeAg峰值分别出现在第1天、第1周、第4周和第10周。对照组HBsAg和HBeAg表达分别呈现2个或3个明显的峰:HBsAg峰值分别出现在第3天和第8周;HBeAg峰值分别出现在第1天、第3周和第10周。空白组未检测出HBsAg和HBeAg。实验组HBV DNA拷贝数高于对照组的拷贝数(P<0.01);肝组织中HBV DNA拷贝数高于同期血清中的拷贝数(P<0.01);实验组和对照组的肝组织中均有HBsAg和HBcAg的表达;实验组与对照组出现肝脏细胞炎症、肝细胞纤维化、肝细胞坏死等病理变化,而空白组正常。结论 利用水动力法向C57BL/6小鼠体内转入HBV cccDNA,成功建立了慢性乙型肝炎病毒感染的小鼠模型,与对照组比较,新建立的小鼠乙肝模型具有更高的HBV表达,动物模型为研究乙型肝炎病毒HBV cccDNA的感染及其引起肝损伤的机制奠定了基础。
英文摘要:Objective To establish the mouse model of chronic viral hepatitis by hydrodynamic injection of HBV cccDNA in C57BL/6 mice. Methods A total of 29 C57BL/6 mice were divided into experimental group, control group and blank group, and injected with HBV cccDNA, pAAV-HBV1.2 and isotonic saline by hydrodynamic method, respectively. The serum samples and liver tissues were collected at different time points after injection. Radioimmunoassay was used to examine the levels of HBsAg and HBeAg. Real time PCR was used to determine the copy numbers of HBV DNA in serum and the liver. Immunohistochemistry was used to examine the levels of HBsAg and HBcAg in liver. HE staining was used to check the pathological changes of liver. SPSS software (version 17.0) was used to analyze the experimental data. Results The expression level of HBsAg in the experimental group showed a pattern of 4 peaks at day 3 and week 3, week 7 and week 9. A similar 4-peak expression pattern was detected in HBeAg at day 1 and week 1, week 4 and week 10. In the control groups, only 2 peaks at day 3 and week 8 were found in the expression pattern of HbsAg, and the peak value of HBeAg was observed at day 1, week 3, and week 10. No expression of HBsAg and HBeAg was detected in the blank group. As determined by real time PCR, although the levels of HBV DNA decreased with time, the copy numbers were significantly higher in the experimental group than those of control (P<0.01). Moreover, the copy numbers of HBV DNA in mice liver were significantly higher than those in the serum (P<0.01) at all time points. Positive expression of HBsAg and HBeAg in liver was revealed by immunohistochemistry in the experimental and control groups. The result of HE staining confirmed the symptoms of inflammation, fibrosis and necrosis in mice liver tissues. Conclusion HBV chronic infection model in C57BL/6 mice was successfully established by in vivo transduction with HBV cccDNA. The expression of HBV in the experimental groups was higher than that of control. This study provided a basic model for further understanding the mechanism of HBV infection and liver injury.
2015,34(2): 200-207 收稿日期:2014-6-23
DOI:10.3969/j.issn.1000-7083.2015.02.007
分类号:Q95-33;R512.6
基金项目:重庆市科委重大项目(CSTC2013yykfc1005)
作者简介:严磊(1985—),男,硕士研究生,研究方向为实验动物学,E-mail:985466685@qq.com
*通讯作者:赖国旗,E-mail:a68895078@21cn.com
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