高压水动力法建立表面抗原表达缺失的乙型肝炎转染小鼠模型及初步评估

Establishment and preliminary evaluation of HBsAg mutated hepatitis B transfected mice model viahydrodynamic injection

目的探讨HBsAg在小鼠体内模型中对宿主免疫应答调节及病毒感染慢性化中的作用。方法在已有高压水动力法转染pAAV-HBV1.2质粒建立乙肝小鼠模型的基础上,通过点突变技术引入终止密码子至pAAV-HBV1.2质粒上的HBsAg读码框,构建HBsAg无法转录的突变质粒,再利用该质粒构建HBsAg表达缺失的乙肝小鼠模型。通过比较原始质粒建立的乙肝模型小鼠,检测分析外周血中HBV抗原与DNA等病毒感染指标,比较肝脏病变和免疫细胞浸润情况。结果成功构建了HBsAg缺失的乙肝模型小鼠:在乙肝模型小鼠和HBsAg缺失突变的模型小鼠外周血中均能检测到4 COI/mL水平的HBeAg,两组小鼠没有差异,但只有乙肝小鼠体内能检测到102 COI/mL以上的HBsAg和103至106拷贝/mL的HBV DNA,HBsAg缺失的小鼠中无法检测到HBsAg和HBV DNA;动态监测显示HBeAg在乙肝和HBsAg缺失突变的两组小鼠中的阳性率均逐步降低,在HBsAg缺失突变组中降低的更明显,第8周时仅有20%的小鼠为HBeAg阳性,而此时未突变乙肝组的阳性率为60%;免疫组织化学结果显示:在阴性对照小鼠的肝脏结构较完整,未见聚集的浸润免疫细胞,而乙肝和HBsAg缺失的乙肝小鼠肝小叶结构均松散,乙肝小鼠肝脏中有聚集的浸润性免疫细胞,HBsAg缺失的小鼠肝脏缺少免疫细胞的聚集,却存在严重的肝细胞毛玻璃样变性。结论成功构建了HBsAg缺失的乙肝转染小鼠模型;并发现HBsAg在HBV完整病毒颗粒包装和出胞,以及肝脏病变和免疫细胞浸润过程中发挥了重要的作用。

Objective To investigate the role of hepatitis B surface antigen（HBsAg）in chronicity of hepatitis B virus（HBV）and regulation of host immune responses in vivo.Methods Site-directed mutagenesis was used to establish HBsAg mutated HBV mice model,based on transfection of plasmid adeno-associated virus（pAAV）-HBV through hydrodynamic injection.Then the serum HBsAg,hepatitis B e antigen（HBeAg）and HBV DNA were measured to evaluate the infection in both mice models.In addition,hematoxylin-eosin（HE）staining was performed to analyze pathological changes and infiltration of immune cells in liver.Results HBsAg mutated HBV mice model was successfully established.HBeAg levels in HBV mice model and HBsAg mutated HBV mice model were both 4 COI/ml with no difference.HBsAg and HBV DNA were undetected in HBsAg mutated HBV mice model,while HBsAg was 〉10^2 COI/ml and HBV DNA was 10^3- 10^6 copies/ml in HBV mice model.Dynamic monitor showed that percentage of HBeAg positive rate gradually declined in both mice models,which was more obvious in HBsAg mutated HBV model.At week 8,HBeAg positive rate was 20% in HBsAg mutated HBV model compared to 60%in HBV model.Compared with HBV model,there was less infiltration of immune cells but more ground-glass hepatocytes were in the liver of HBsAg mutated HBV mice model with HBsAg deficiency.Conclusion HBsAg mutated HBV mice model was successfully established.HBsAg might have an important role in pathologic process and infiltration of immune cells in liver.It lays a foundation for further study on the role of HBsAg in HBV replication and immunologic escape.