甲型 H1N1 流感病毒感染年轻和老年 C57BL / 6 小鼠诱导肺组织 CD8+ T 细胞特异性免疫应答的比较研究
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复旦大学附属公共卫生临床中心,上海 201508


Study of the immune responses mediated by specific CD8+T cells to influenza A virus H1N1 in the lungs of young and aged C57BL/ 6 mice
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Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China

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    目的 比较甲型 H1N1 流感病毒 PR8 毒株感染老年和年轻 C57BL/ 6 小鼠诱导肺组织 CD8+ T 细胞特异性免疫应答的能力。 方法 使用 490 PFU 的 PR8 病毒分别滴鼻感染年轻(3 月龄)和老年(24 月龄)C57BL/ 6 小鼠,连续 14 d 每日记录其体重变化及死亡情况。在感染后第8天分离小鼠肺组织细胞,使用流式细胞术 (fluorescence-activated cell sorting,FACS)技术检测病毒抗原特异性 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 细胞经流感病毒特异性肽刺激后分泌细胞因子水平包括 TNF-α、IFN-γ、IL-2 以及与 CD8+ T 细胞杀伤功能有关的颗粒酶 B(Granzyme B)的水平。 结果 相同量的 PR8 流感病毒感染小鼠后,老年小鼠体重下降更多,死亡率显著高于年轻小鼠(P< 0. 01)。 另一方面,老年组小鼠诱生的病毒特异性 CD8+T 细胞比例显著低于年轻组;同时, 老年组 CD8+T 细胞活化后分泌细胞因子 TNF-α、IFN-γ、IL-2 的水平显著低于年轻组;此外,老年组 CD8+T 细胞表达 granzyme B 的水平同样显著低于年轻组。 结论 PR8 流感病毒感染老年和年轻 C57BL/ 6 小鼠后,老年小鼠肺组织诱导产生的特异性 CD8+T 细胞的数量减少且功能降低。结果表明:与年轻小鼠相比,老年小鼠肺组织 CD8+ T 细胞特异性免疫应答功能受损。

    Abstract:

    Objective To compare the immune response of specific CD8+ T cells induced by H1N1 Influenza A virus PR8 strain in the lungs of young and aged C57BL/ 6 mice. Methods Young ( 3 months) and aged ( 24 months) mice were intranasally infected with 490 plaque-forming units of PR8, then 8 days later, they were euthanized and their lungs collected. Fluorescence-activated cell sorting was used to compare the number and functions of viral antigen-specific CD8+T cells between the young and aged mice. Surface staining for the influenza virus MHC-I tetramer was performed to count the antigen-specific CD8+T cells; and intracellular cytokine staining was used to quantify the cytokines secreted by CD8+ T cells in response to influenza-specific peptides, including tumor necrosis factor ( TNF-α ), interferon-γ, interleukin-2, and granzyme B, which are involved in killing by CD8+ T cells.Results The proportion of virus-specific CD8+T cells in the aged mice was significantly lower than in the young mice. Moreover, the expression of interferon-γ, TNF-α, and interleukin-2 in activated CD8+T cells from the aged mice was significantly lower than in the young mice. In addition, the level of granzyme B expression on CD8+T cells in the aged mice was significantly lower. Conclusions The number of specific CD8+T cells induced by PR8 influenza virus was lower, and their function was impaired in the lungs of aged C57BL/ 6 mice compared with young mice. This implies that the response by specific CD8+T cells in the lungs of mice is impaired by aging.

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刘洋,王超,任晓楠,李顺,秦波音,杨华,周晓辉.甲型 H1N1 流感病毒感染年轻和老年 C57BL / 6 小鼠诱导肺组织 CD8+ T 细胞特异性免疫应答的比较研究[J].中国实验动物学报,2021,29(4):461~466.

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  • 收稿日期:2021-06-15
  • 在线发布日期: 2021-09-10
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