重症甲型H1N1流感病毒感染诱导小鼠胸腺凋亡萎缩

Severe influenza A(H1N1)virus infection induces thymic atrophy in mice

目的:探讨重症流感病毒感染介导的胸腺萎缩及其作用机制。方法:4~6周龄SPF级BALB/c小鼠滴鼻给予50μl TCID50甲型H1N1流感病毒A/California/07/2009(1×10^6个/ml),构建重症流感病毒感染小鼠模型;检测各感染时间小鼠体质量、胸腺大小及重量;HE染色检测胸腺组织病理变化;流式细胞术检测胸腺内不同发育阶段T细胞数量及凋亡;原位TUNEL法检测胸腺细胞凋亡;细胞凋亡相关基因芯片和荧光定量PCR检测验证促凋亡和抗凋亡相关基因变化。结果:成功建立小鼠重症甲型H1N1流感病毒感染模型。感染2~3 d后小鼠胸腺萎缩变小,以胸腺皮质区萎缩为主,感染5~7 d皮质区完全消失。主要存在于皮质区的双阳性T细胞(CD4^+CD8^+),随感染过程进展逐渐减少直至消失。细胞凋亡,尤其是皮质区细胞凋亡增多是胸腺萎缩的重要原因。促凋亡相关基因表达增加,尤其Fas介导的细胞凋亡可能是胸腺皮质区细胞凋亡的重要机制。结论:重症流感病毒感染可诱导胸腺(尤其是皮质区)萎缩,FasL-Fas介导的细胞凋亡是胸腺萎缩的重要原因。

Objective:To investigate thymic atrophy mediated by severe infection of influenza virus,and its mechanisms.Methods:4-6 weeks of SPF grade BALB/c mice were inoculated intranasally with 50μl of TCID50 influenza A H1N1 influenza virus A/California/07/2009(1×10^6 cells/ml)to establish a mouse model of severe influenza virus infection.Body weight,thymus size and weight at different days post infection were detected;HE staining was used to detect pathological changes of thymus;Flow cytometry was used to detect proportion and apoptosis of T cells in different developmental stages in thymus.In situ TUNEL method was used to detect apoptosis of thymocytes.Apoptosis-related gene chip and real-time PCR to confirm changes of pro-apoptotic and anti-apoptosis-related genes after infection.Results:A mouse model of severe influenza A(H1N1)infection was successfully established.Thymus of infected mice became smaller at 2-3 d post infection,and atrophy of thymic cortex was dominant until cortical area disappeared completely at 5-7 d post infection.Double positive(CD4^+CD8^+)T cells,which mainly present in cortical area,gradually decreased and until disappeared as infection progressed.Apoptosis,especially in cortical region is an important cause of thymus atrophy.Expressions of pro-apoptosis-related genes were increased,especially Fas-mediated apoptosis may be an important mechanism of apoptosis in thymic cortex.Conclusion:Severe influenza virus infection can induce atrophy of thymus(especially cortical area),and FasL-Fas-mediated apoptosis is an important cause of thymus atrophy.