免疫功能正常的HBV cccDNA小鼠模型用于苦参碱和青蒿素抑制HBV的药效评价

doi:10.12300/j.issn.1674-5817.2021.016

实验动物与比较医学 ›› 2021, Vol. 41 ›› Issue (2): 108-115.DOI: 10.12300/j.issn.1674-5817.2021.016

所属专题：期刊发展

• 论著:人类疾病动物模型 • 上一篇下一篇

免疫功能正常的HBV cccDNA小鼠模型用于苦参碱和青蒿素抑制HBV的药效评价

伍悦¹, 吕小琴², 刘阳^2,3, 向霞², 赵中华⁴, 徐如青², 潘朔寒², 何明忠², 张华堂⁴, 赖国旗²

1.重庆医科大学附属第二医院,重庆 400010;
2.重庆医科大学实验动物中心,重庆 400016;
3.泸县第二中学,泸州 646106;
4. 重庆市科学技术研究院,重庆 401123

收稿日期:2021-01-16 修回日期:2021-03-11 出版日期:2021-04-25 发布日期:2021-04-30
作者简介:伍悦(1992—), 男, 硕士研究生, 研究方向: 肝胆外科。E-mail: truth1105@qq.com
基金资助:
国家自然科学基金资助项目(81570541); 重庆市科委社会民生项目(cstc2017shmsA130097); 重庆医科大学大学生研究与创新实验项目(SRIEP202049)

Application of Immunocompetent HBV cccDNA Mouse Model in the Efficacy Evaluation of Matrine and Artemisinin on Inhibiting HBV

WU Yue¹, LÜ Xiaoqin², LIU Yang^2,3, XIANG Xia², ZHAO Zhonghua⁴, XU Ruqing², PAN Shuohan², HE Mingzhong², ZHANG Huatang⁴, LAI Guoqi²

1. Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China;
2. Laboratory Animal Center of Chongqing Medical University, Chongqing 400016, China;
3. Luxian No.2 High School, Luzhou 646106, China;
4. Chongqing Academy of Science and Technology, Chongqing 401123, China

Received:2021-01-16 Revised:2021-03-11 Online:2021-04-25 Published:2021-04-30

摘要/Abstract

摘要： 目的应用免疫功能正常的乙型肝炎病毒（hepatitis B virus,HBV）小鼠模型评价中药苦参碱和青蒿素的作用效果,同时为模型应用奠定基础。方法体外合成HBV共价闭合环状DNA（covalently closed circular DNA,cccDNA）,通过尾静脉高压注入8~10周龄的雄性CBA/CaJ小鼠体内,然后分别在急性感染期和慢性感染期腹腔注射苦参碱或青蒿素。定量PCR法检测小鼠血清及肝组织中HBV DNA和cccDNA含量,酶联免疫吸附法检测血清中HBV表面抗原（HBV surface antigen,HBsAg）的含量,免疫组织化学法检测肝组织中HBsAg和HBV核心抗原（HBV core antigen,HBcAg）的表达。结果在HBV急性感染期,注射苦参碱和青蒿素第4周即能够明显抑制小鼠血清中HBsAg的分泌（均P<0.05）,降低血清中HBV DNA的表达（均P<0.01）。在HBV慢性感染期,苦参碱和青蒿素对小鼠血清和组织中HBsAg的抑制作用不明显（均P>0.05）,但是血清中HBsAg相对降低水平（∆HBsAg）明显大于生理盐水对照组（均P<0.05）;而且苦参碱能降低小鼠血清或组织中HBV DNA（P<0.05,P<0.01）和组织中HBV cccDNA的拷贝数（P<0.05）;青蒿素仅能明显抑制小鼠血清中HBV DNA拷贝数（P<0.01）,而对组织中HBV DNA和HBV cccDNA的作用不明显（均P>0.05）;另外,苦参碱对肝组织中HBcAg的作用不明显（P>0.05）,而青蒿素对HBcAg有明显的抑制作用（P<0.05）。结论免疫功能正常的HBV cccDNA小鼠模型能够用于治疗急性期和慢性期乙型肝炎的药物疗效评价。

关键词: 乙型肝炎病毒, 慢性感染, 药效评价, 苦参碱, 青蒿素, CBA/CaJ小鼠

Abstract: Objective To evaluate the effects of matrine and artemisinin on immunocompetent hepatitis B virus (HBV) infected mouse model, and to lay a foundation for the application of the model. Methods HBV covalently closed circular DNA (HBV cccDNA) was synthesized in vitro. Male CBA/CaJ mice aged 8 to 10 weeks were injected with HBV cccDNA at high pressure through tail vein, and then intraperitoneally injected with matrine or artemisinin in acute or chronic infection periods, respectively. Quantitative PCR was used to detect HBV DNA and cccDNA in serum or liver tissues of mice. Enzyme-linked immunoadsorption assay (ELISA) was used to measure the content of HBV surface antigen (HBsAg) in serum. Immunohistochemistry was used to determine HBsAg and HBV core antigen (HBcAg) in liver tissues. Results In HBV acute infection period, matrine and artemisinin injection at week 4 significantly inhibited the secretion of HBsAg in serum of mice (both P<0.05), and reduced the expressions of HBV DNA (both P<0.01). In HBV chronic infection period, matrine and artemisinin had no significant inhibitory effects on HBsAg in serum and liver tissues (all P>0.05), but the decrease of HBsAg in serum in the group administered with matrine or artemisinin was significantly higher than that in the normal saline control group (both P<0.05). Moreover, matrine decreased the copy number of HBV DNA in serum and liver tissues (P<0.05 and P<0.01, respectively) and the copy number of HBV cccDNA in liver tissues (P<0.05). Artemisinin only inhibited the copy number of HBV DNA in serum of mice (P<0.01), but had no significant effect on HBV DNA and HBV cccDNA in liver tissues (both P>0.05). In addition, matrine had no significant effect on HBcAg in liver tissues (P>0.05), but artemisinin did the opposite (P<0.05). Conclusion The immunocompetent HBV cccDNA mouse model can be used to evaluate the efficacy of drugs for the treatment of acute and chronic hepatitis B.

Key words: Hepatitis B virus, Chronic infection, Efficacy evaluation, Matrine, Artemisinin, CBA/CaJ mice

中图分类号:

R-332
Q95-33

伍悦, 吕小琴, 刘阳, 向霞, 赵中华, 徐如青, 潘朔寒, 何明忠, 张华堂, 赖国旗. 免疫功能正常的HBV cccDNA小鼠模型用于苦参碱和青蒿素抑制HBV的药效评价[J]. 实验动物与比较医学, 2021, 41(2): 108-115.

WU Yue, LÜ Xiaoqin, LIU Yang, XIANG Xia, ZHAO Zhonghua, XU Ruqing, PAN Shuohan, HE Mingzhong, ZHANG Huatang, LAI Guoqi. Application of Immunocompetent HBV cccDNA Mouse Model in the Efficacy Evaluation of Matrine and Artemisinin on Inhibiting HBV[J]. Laboratory Animal and Comparative Medicine, 2021, 41(2): 108-115.

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免疫功能正常的HBV cccDNA小鼠模型用于苦参碱和青蒿素抑制HBV的药效评价

Application of Immunocompetent HBV cccDNA Mouse Model in the Efficacy Evaluation of Matrine and Artemisinin on Inhibiting HBV

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