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秦波音,王超,刘洋,谭丹,方钟,李顺,周晓辉.受体相互作用蛋白激酶-3 参与甲型流感病毒H1N1感染C57BL/ 6 小鼠诱导特异性CD8+ T 细胞应答[J].中国实验动物学报,2019,27(5):549~554.
受体相互作用蛋白激酶-3 参与甲型流感病毒H1N1感染C57BL/ 6 小鼠诱导特异性CD8+ T 细胞应答
Receptor-interacting protein 3 plays a role in the induction of influenza viral antigen specific CD8+T cell responses in C57BL / 6 mice upon H1N1 influenza virus infection
投稿时间:2019-08-05  
DOI:10. 3969 / j.issn.1005-4847. 2019. 05. 001
中文关键词:  RIP3  流感病毒  特异性CD8+ T 细胞
英文关键词:RIP3  influenza virus  influenza-Specific CD8+ T cells
基金项目:
作者单位E-mail
秦波音 复旦大学附属公共卫生临床中心,上海 201508 qinboyin@ shphc.org.cn 
王超 复旦大学附属公共卫生临床中心,上海 201508  
刘洋 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  
方钟 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  
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中文摘要:
      目的 研究受体相互作用蛋白激酶-3(receptor-interacting protein 3, RIP3)在C57BL/6 小鼠感染甲型流感病毒H1N1 PR8 后,在特异性CD8+ T 细胞免疫应答中的作用?方法 用剂量为0. 7 × 103 半数组织细胞感染剂量(50% tissue culture infective dose, TCID50)的甲型流感H1N1 PR8 病毒株,通过滴鼻方式分别感染RIP3 敲除(RIP3-/ - )和野生型(WT)的C57BL/6 小鼠?在感染后第8 天(day post infection, d.p.i)分离小鼠脾细胞,使用流式细胞术(fluorescence-activated cell sorting, FACS)检测流感抗原特异性CD8+ T 细胞数量及功能:MHC I 表位肽四聚体法(tetramer staining)染色流感特异性CD8+ T 细胞,细胞内细胞因子染色法(intracellular cytokine staining, ICS)检测CD8+ T 细胞产生IFN-γ?TNF-α?IL-2 等效应性细胞因子水平和与CD8+ T 细胞杀伤功能有关的颗粒酶(granzymeB)的表达情况?结果 0. 7 × 103 TCID50流感病毒感染后,WT 小鼠中流感抗原特异性CD8+ T 细胞比例为RIP3-/ -小鼠的2. 71 倍;WT 小鼠中CD8+ T 细胞分泌细胞因子IFN-γ?TNF-α?IL-2 的能力均显著高于RIP3-/ - 小鼠( P <0. 01);且WT 小鼠CD8+ T 表达granzyme B 水平显著高于RIP3-/ -小鼠( P < 0. 01)?结论 本研究在甲型流感病毒感染的小鼠体内发现敲除RIP3 分子可导致流感感染诱导的特异性CD8+ T 细胞数量减少?功能降低,表明RIP3 是参与流感抗原特异性CD8+ T 细胞的诱生及功能发挥的关键分子之一?
英文摘要:
      Objective To investigate whether RIP3 (receptor-interacting protein 3) is involved in the inductionof influenza antigen specific CD8+ T cell responses in C57BL/6 mice upon H1N1 influenza virus infection. Methods RIP3knockout (RIP3-/ - ) and wild type (WT) C57BL/6 mice were infected by influenza virus H1N1 PR8 with the dose of 0. 7×103 TCID50(50% tissue culture infective dose). Splenocytes were isolated from the infected mice which were sacrificed at 8days post infection (d.p.i), and fluorescence-activated cell sorting (FACS) were used for the comparison of quantities andfunctions of influenza viral antigen specific CD8+ T cells between the RIP3-/ - group and WT group. Surface staining ofinfluenza viral MHC I tetramer was performed to quantify the antigen specific CD8+ T cells. Intracellular cytokine staining(ICS) was carried out to dissect the production levels of effector cytokines, such as IFN-γ, TNF-α and IL-2. Granzyme B,which is related to the cytotoxic effect of the antigen specific CD8+ T cells, was also assayed by ICS. Results On average,the percentages of influenza viral antigen specific CD8+ T cells in the WT group of mice were about 2. 71 fold comparingwith that in the RIP3-/ - group of mice. Furthermore, the production levels of IFN-γ, TNF-α and IL-2 by the antigen specificCD8+ T cells in the WT group of mice were significantly higher than those levels in the counterpart group of RIP3-/ - mice( P < 0. 01); and in regards to granzyme B, the same trend was showed when comparing the expression levels between thosetwo group mice, i.e., WT group was higher than the RIP3-/ - group ( P < 0. 01). Conclusions The data of the presentstudy reported that the deficiency of RIP3 molecule in C57BL/6 mice will decrease the quantity and functionality of theinfluenza viral antigen specific CD8+ T cells at 8 d.p.i. upon influenza virus H1N1 PR8 infection, which indicates that RIP3 is indispensable for the induction of influenza viral antigen specific CD8+ T cells at effector stage.
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