摘要
目的 探索HBV感染所致肝纤维化小鼠模型形成条件及表型分析。方法 以18只CBA/CaJ小鼠为研究对象,随机分为对照组(n=4)和实验组(n=14),实验组按照每只2μg的剂量、通过高压水动力方法进行尾静脉注射cccDNA,对照组注射等体积PBS,转染68周后采样。通过qPCR方法检测肝中HBV DNA及HBV cccDNA,ELISA方法检测血清HBsAg、HBeAg,免疫组化检测肝组织中HBsAg、HBcAg,用一代测序法检测HBeAg+/HBsAg-及HBeAg-/HBsAg+组织DNA基因测序,生化分析ALT和AST,HE染色、天狼星染色及Masson染色分析肝组织病理。结果 实验组中,100%的小鼠HBV-DNA拷贝数高于1000 copies/mL;小鼠肝组织cccDNA拷贝数显著高于空白组(P<0.05);42.9%的小鼠血清HBsAg和HBeAg呈阳性;60%的小鼠肝HBsAg呈阳性和26.7%的小鼠肝HBcAg呈阳性;HBeAg-/HBsAg+样本中出现A1762T/G1764A和G1896A/G1899A位点突变,HBeAg+/HBsAg-样本出现前S2区及S区多位点突变;小鼠ALT值异常率为57.1%,AST值异常率为7.1%;HE染色观察到所以实验组小鼠肝组织损伤及肝炎症浸润,天狼星染色、Masson染色观察到92.9%的肝组织发生纤维化。结论 通过尾静脉高压水动力注射CBA/CaJ小鼠HBV cccDNA 68周后,HBV主要DNA、HBV cccDNA、血清及肝组织HBsAg、HBeAg仍然阳性,HBV感染可引起小鼠肝纤维化,甚至可模拟临床HBV BCP/PC基因突变表型。
Objective To explore the formation conditions and phenotype of a mouse model of liver fibrosis induced by HBV(hepatitis B virus,HBV)infection.Methods Eighteen CBA/CaJ mice were randomly divided into control group(n=4)and experimental group(n=14),and the experimental group was injected with cccDNA by tail vein at a dose of 2μg per mouse by high⁃pressure hydrodynamic method,and the control group was injected with an equal volume of PBS.Samples were obtained 68 weeks after transfection.HBV DNA and HBV cccDNA in mouse livers were detected by quantitative PCR.Serum HBsAg(hepatitis B surface antigen,HBsAg)and HBeAg(hepatitis Be antigen,HBeAg)levels were tested using ELISA.HBsAg and HBcAg liver tissue levels were tested by immunohistochemistry.HBV DNA in mouse liver tissue,with the phenotype HBeAg+/HBsAg-or HBeAg-/HBsAg+,were first⁃generation sequenced.ALT and AST levels were evaluated by biochemical analysis,and histopathology was analyzed by HE,Sirius,and Masson staining.Results In the experimental group,100%of mouse HBV DNA was higher than 1000 copies/mL.The cccDNA copy number of mouse liver tissue was significantly higher than that of the blank group(P<0.05);42.9%of mice were positive for HBsAg and HBeAg,60%were positive for HBsAg,and 26.7%were positive for HBcAg.Mutations at A1762T/G1764A and G1896A/G1899A were found in HBeAg-/HBsAg+samples,and there were multiple mutations in the S2 and S regions of HBeAg+/HBsAg-samples.ALT and AST values were abnormal in 57.1%and 7.1%of mice,respectively.Liver damage and infiltration were observed in all experimental mice,and liver tissue fibrosis was found in 92.9%of mice.Conclusions HBV DNA、HBV cccDNA、HBsAg、HBeAg of Serum and liver tissue remained positive 68 weeks after hydrodynamically injecting HBV cccDNA into CBA/CaJ mice.HBV infection caused liver fibrosis in mice and even mimicked a clinical HBV BCP/PC gene mutation phenotype.
作者
何苗
向霞
徐如青
伍悦
吕小琴
刘阳
何明忠
赖国旗
HE Miao;XIANG Xia;XU Ruqing;WU Yue;LYU Xiaoqin;LIU Yang;HE Mingzhong;LAI Guoqi(Laboratory Animal Center of Chongqing Medical University,Chongqing 400016;China.2.First Affiliated Hospital of Chongqing Medical University,Chongqing 400042)
出处
《中国实验动物学报》
CAS
CSCD
北大核心
2023年第2期187-193,共7页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金(81570541)
重庆市科委社会民生项目(cstc2017shmsA130097)。
