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王超,刘洋,秦波音,任晓楠,谭丹,杨华,李顺,周晓辉.甲型流感病毒H1N1 pdm09 与PR8 感染C57BL/ 6 小鼠诱生特异性CD8+ T细胞免疫应答的比较研究[J].中国实验动物学报,2019,27(5):561~570.
甲型流感病毒H1N1 pdm09 与PR8 感染C57BL/ 6 小鼠诱生特异性CD8+ T细胞免疫应答的比较研究
Comparative study on immune responses of specific CD8+T cells induced by influenza A virus H1N1 PR8 and pdm09 in C57BL / 6 mice
投稿时间:2019-08-05  
DOI:10. 3969 / j.issn.1005-4847. 2019. 05. 003
中文关键词:  甲型流感病毒  特异性CD8+ T 细胞  免疫应答  功能耗竭
英文关键词:influenza A virus  specific CD8+ T cells  immune response  functional depletion
基金项目:
作者单位E-mail
王超 复旦大学附属公共卫生临床中心,上海 201508 584264751@ qq.com 
刘洋 复旦大学附属公共卫生临床中心,上海 201508  
秦波音 复旦大学附属公共卫生临床中心,上海 201508  
任晓楠 复旦大学附属公共卫生临床中心,上海 201508  
谭丹 复旦大学附属公共卫生临床中心,上海 201508  
杨华 复旦大学附属公共卫生临床中心,上海 201508  
李顺 复旦大学附属公共卫生临床中心,上海 201508  
周晓辉 复旦大学附属公共卫生临床中心,上海 201508 zhouxiaohui@ shphc.org.cn 
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中文摘要:
      目的 比较甲型流感病毒H1N1 两亚型毒株A/ Shanghai/37T/2009(H1N1)(pdm09)临床分离株和A/ Puerto Rico/8/34(H1N1)(PR8)标准株感染C57BL/6 小鼠后,其病理状态的改变及诱生特异性CD8+ T 细胞的免疫反应?方法 ①用9. 85 × 106 半数组织细胞感染剂量(50% tissue culture infective dose,TCID50)的PR8 和pdm09病毒分别滴鼻感染野生型C57BL/6 小鼠,每日(连续14 d)观察其状态并记录死亡情况?于感染后第1?3 天处死小鼠,取其肺称重计算肺指数;左叶肺经4%多聚甲醛固定后进行H&E(hematoxylin-eosin staining)染色,观察感染后肺的病理变化;实时荧光定量PCR(qRT-PCR)检测肺病毒载量?② 用0. 25 倍半数致死量(lethal dose 50%,LD50)的PR8 和pdm09 分别滴鼻感染小鼠,每日称重连续观察?于感染后第8 天解剖取肺和脾,通过H&E 染色比较肺部病理改变;通过流式细胞术(FACS)检测脾病毒特异性CD8+ T 细胞数量比例,以及IFN-γ?TNF-α?IL-2?granzyme B 等细胞因子和免疫抑制检查点分子PD-1?LAG-3?Tim-3?CTLA-4 的表达情况?结果 ①以相同TCID50 流感病毒感染后,两组小鼠均出现疾病状态?PR8 组小鼠感染第5 天全部死亡,死亡率显著高于pdm09 组( P < 0. 01);第1?3 天肺指数和病毒载量的结果PR8 组均显著高于pdm09 组;H&E 染色结果显示PR8 组病理损伤严重,肺泡壁增厚,出现肺实质化,血细胞渗出增多,而pdm09 组小鼠肺组织病理损伤较轻,炎症浸润少;② 以相同LD50 流感病毒感染后,PR8 组小鼠的体重损失和肺组织病理损伤均比pdm09 组严重;流式结果显示PR8 组诱生的病毒特异性CD8+ T细胞比例高于pdm09 组,但是PR8 组中活化的CD8+ T 细胞(CD8+ CD44High )表达的IFN-γ?IL-2 和TNF-α 显著低于pdm09 组;检测该群细胞免疫功能耗竭分子,发现其表达了更多的免疫抑制分子PD-1 和LAG-3?结论 PR8 相对于pdm09 能够对小鼠造成更严重的损伤,可能是因为其活化的特异性的CD8+ T 细胞出现功能耗竭导致特异的免疫保护力受损,抵抗和清除病毒的能力下降,故而造成更严重的病理损伤?
英文摘要:
      Objective To compare the pathogenicity of two subtypes of influenza A virus A/ Shanghai/37T/2009(H1N1) (pdm09) and A/ PuertoRico/8/34 (H1N1) (PR8) and the immunogenicity of those two subtypes of influenza Avirus to induce antigen specific CD8+ T cells in C57BL/6 mice. Methods ①The wild type C57BL/6 mice were infectedwith 50% tissue infection dose (TCID50) of PR8 and pdm09 respectively. The situations of the infected mice were observeddaily (14 days), and the death rate was recorded. The mice were sacrificed at 1 and 3 days (days post infection, d.p.i),the lungs were collected to weigh and the lung indexes were calculated, and the left lobes of lung were fixed by 4%paraformaldehyde and stained with H&E to observe the pathological changes of the lung after infection. The viral load inlung tissue was detected by real-time fluorescence quantitative PCR (qRT-PCR). ② The mice were intranasally infectedwith 0. 25 times lethal dose (lethal dose 50%, LD50) of PR8 and pdm09, respectively. The infected mice were weighedevery day and monitored continuously for 14 days. The splenocytes were isolated on the 8 d.p.i. The percentages of influenzaviral antigen specific CD8+ T cells, the expression levels of IFN-γ, TNF-α, IL-2, granzyme B and immunosuppressivecheckpoint molecules PD-1, LAG-3, Tim-3 and CTLA-4 were detected by fluorescence-activated cell sorting (FACS).Results ①After the same TCID50 influenza virus infections, the mice in both groups were morbid. All the mice in thePR8 group died on the 5th day after infection, and the mortality rate in the PR8 group was significantly higher than thatin the pdm09 group on the 1st and 3rd day ( P <0. 01), and the lung index and viral load on the 1st and 3rd day in thePR8 group were significantly higher than those in the pdm09 group. The result of H&E staining showed that thepathological injury of lung tissue was mild and the inflammatory infiltration was less in the pdm09 group. ② After thesame LD50 influenza virus infection, the weight-loss and pathological changes in the lung tissues in the PR8 group weresignificantly more serious than in the pdm09 group. The result of flow cytometry showed that the proportion of virusspecificCD8+ T cells in the PR8 group was higher than that in the pdm09 group, but the productions of IFN-γ, TNF-αand IL-2 by activated CD8+ T cells in the PR8 group were significantly lower than that in pdm09 group. Further detectionof exhaustion makers on the activated CD8+ T cells revealed that the cells of the PR8 group express moreimmunosuppressive molecules PD-1 and LAG-3 than those cells of the pdm09 group. Conclusions Influenza A virusPR8 can cause more serious damage in mice compared with pdm09, which may be due to the functional exhaustion ofactivated antigen specific CD8+ T cells, resultsing in lower protective immunity against influenza viral infection and more severe pathological injury than pdm09 infection.
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