【摘要】 目的:通过构建急性乙型肝炎病毒(HBV)感染小鼠模型,观察肝脏内γδT细胞的比例、激活表型及细胞因子表达的变化,探讨γδT细胞在HBV急性感染清除机制中的作用。方法:用尾静脉高压水动力法注射HBV基因组1.3倍体真核表达质粒(pcDNA3.1-HBV1.3)构建急性乙型肝炎病毒感染小鼠模型,用流式细胞仪方法检测pcDNA3.1-HBV1.3质粒模型组(pHBV组)、pcDNA3.1对照质粒组(pcDNA组)在质粒注射后不同时间肝脏、脾脏及外周血中γδT细胞比例的变化,以及pHBV组肝脏中γδT细胞的CD25与CD69激活分子,IFN-γ及TNF-α细胞因子产生的变化,用SPSS统计软件分析各时间点及各组间的差异。结果:pHBV组血清中的HBsAg在第1天即为阳性(33.64±14.88 COI),到第5天上升到最高(146.92±24.23 COI),而后逐渐降低;而血清中的HBeAg在第1天为阳性并达峰值(2.26±2.34 COI),然后降低。而正常对照小鼠(NC)与pcDNA组的外周血中HBV标志物一直为阴性。在pHBV组中,肝脏内的γδT细胞的比例在第1天即升高至(8.3±4.3)%,而后降低,到第10天时,又快速升高至(8.0±1.2)%。统计结果显示,第1天的比例明显高于第5及15天的比例,差异有显著性(P<0.05)。但各时间点脾脏或外周血中的γδT细胞比例未见有明显的变化,差异无显著性(P>0.05)。在pcDNA组中,小鼠肝脏、脾脏或外周血中的γδT细胞比例均未见有显著性的变化发生(P>0.05)。同时pHBV组小鼠肝脏内CD25+γδT细胞比例在第5天升高至最高(8.4±3.7)%,但各时间点间的差异无显著性(P>0.05);而CD69+γδT细胞比例随着时间延长而逐渐降低,第10天的CD69+γδT细胞比例与其他组比较差异有显著性(P<0.05)。pHBV组第1天IFN-γ+γδT细胞比例升高至(8.6±3.2)%,之后即刻降低,与其他组比较,差异无显著性(P>0.05)。结论:急性乙型肝炎病毒感染小鼠模型构建成功,模型中肝脏内γδT细胞的比例、激活表型或细胞因子产生均发生了显著性变化,提示γδT细胞可能参与了急性HBV感染与清除过程。更多还原

【Abstract】 Objective: To observe the change in the proportion,the activation phenotype,or the cytokine expression of liver γδT cells and explore the role of γδT cells in the clearance mechanism of acute HBV infection by construction of murine acute hepatitis model. Methods: The murine acute hepatitis model was constructed by hydrodynamics-based injection of plasmid containing full-length HBV genome( pcDNA3. 1-HBV1. 3),and the proportion of total γδT cells,or CD25+,CD69+,IFN-γ+,or TNF-α+γδT cells in the liver,spleen,or peripheral blood of mouse was detected by FACS at different time point after injection with pcDNA3. 1-HBV1. 3 plas- mid( pHBV group) or pcDNA3. 1 control plasmid( pcDNA group). The data was statistically analyzed by SPSS software to find wheth- er these differences were significant. Results: HBsAg was positive in the serum of pHBV group on the first day after injection,and HB- sAg reached its peak value at 5 day followed by the declination gradually. And for HBeAg,it was positive and got the maximum level at the first day,then its level decreased. All HBsAg or HBeAg levels at each timepoint were negative in normal control mice( NC) or pcDNA group. In pHBV group,the proportion of liver γδT cells increased to( 8. 3 ± 4. 3) % at the first day,then decreased followed by a quick rise to( 8. 0 ± 1. 2) % at the 10th day. The difference in the proportion between first day and the 5th or 15th day was signif- icant( P < 0. 05). But there were no significant differences in the proportion of spleen or blood γδT cells among all the timepoints( P >0. 05). For the pcDNA group,the statistics results showed no significant differences in the proportion of liver,spleen or blood γδT cells( P > 0. 05). Meanwhile,for the pHBV group,the proportion of liver CD25+γδT cells got the peak of( 8. 4 ± 3. 7) %,but no significant differences were found among these timepoints( P > 0. 05),and the proportion of liver IFN-γ+γδT increased to( 8. 6 ± 3. 2) % at first day,which had no significant difference with that at other day. Conclusion: The murine acute hepatitis model has been successfully constructed. There are much changes in the proportion,activation phenotype,or the cytokine producing of liver γδT of this model. All the results show some role of γδT cells in the process of acute HBV infection and its clearance.更多还原