类泛素化蛋白酶8对T细胞活化与分化的影响  

Effect of sentrin⁃specific protease 8 on T cell activation and differentiation

作　　者：徐光磊 靳远晗 张耀林 程道海 张纪岩 曹俊霞 XU Guanglei;JIN Yuanhan;ZHANG Yaolin;CHENG Daohai;ZHANG Jiyan;CAO Junxia(Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院军事认知与脑科学研究所,北京100850

出　　处：《军事医学》2023年第10期735-739,747,共6页Military Medical Sciences

基　　金：国家自然科学基金项目(82201945)。

摘　　要：目的探讨类泛素化蛋白酶8(SENP8)在T细胞活化和分化过程中的作用。方法C57BL/6背景的Senp8基因打靶小鼠(Senp8F/F小鼠,简称WT小鼠)与同背景的Cd4⁃Cre转基因小鼠交配,获得在T细胞中特异性缺失SENP8的小鼠(Senp8F/F;Cd4⁃Cre小鼠,简称KO小鼠),通过鼠尾基因组DNA PCR以及胸腺细胞免疫印迹实验进行基因型鉴定。流式细胞术分析SENP8缺失对稳态T细胞比例和数量的影响,并以李斯特菌尾静脉注射感染WT和KO小鼠,7 d后流式细胞术分析CD8^(+)T细胞活化、扩增、效应因子表达情况。结果PCR鉴定结果揭示,KO小鼠有Cd4⁃Cre基因和纯合的Loxp片段;免疫印迹结果显示,KO小鼠胸腺细胞中的SENP8蛋白表达下降;证明成功构建了T细胞中特异性敲除SENP8的小鼠模型。流式细胞术检测结果表明,CD4^(+)T细胞和CD8^(+)T细胞在稳态WT和KO小鼠脾脏中的比例和数量并无显著差异。李斯特菌感染7 d后,条件性缺失SENP8不影响CD4^(+)T细胞、CD8^(+)T细胞数量增加和活化标志分子CD44表达,也不影响CD4^(+)T细胞、CD8^(+)T细胞中干扰素γ(IFN⁃γ)和颗粒酶B(GzmB)的表达。结论T细胞中缺失SENP8分子不影响T细胞的活化、扩增及李斯特菌感染过程中T细胞的细胞毒作用。Objective To explore the role of sentrin⁃specific protease 8(SENP8)in T cell activation and differentiation.Methods Mice homozygous for an Senp8 conditional allele(Senp8F/F,WT)on the C57BL/6 background were crossed with Cd4⁃Cre transgenic mice to generate a mouse model with T cell⁃specific deficiency of SENP8(Senp8F/F;Cd4⁃Cre,KO).The genotypes were determined via PCR of tail⁃extracted DNA and immunoblotting analysis of thymocytes.Flow cytometry was employed to examine the number of splenic T cells in homeostatic WT and littermate KO mice.Furthermore,Listeria monocytogenes was injected into the tail vein of WT and littermate KO mice.The activation,expansion,and differentiation of CD8^(+)T cells were determined via flow cytometry 7 days later.Results The result of PCR showed Cd4⁃Cre and homozygous Loxp in KO mice and that of immunoblotting revealed reduced SENP8 in thymocytes from KO mice,indicating that a mouse model was established.Flow cytometry demonstrated comparable numbers of splenic CD4^(+)and CD8^(+)T cells in homeostatic WT and littermate KO mice.7 days after Listeria monocytogenesinfection,the expansion of CD4^(+)and CD8^(+)T cells was not affected by T cell⁃specific SENP8 deficiency,so were the upregulation of activation marker CD44 in CD4^(+)and CD8^(+)T cells and the expressions of effector factors interferon⁃γ(IFN⁃γ)and granzyme B(GzmB).Conclusions SENP8 does not affect T cell activation,expansion or cytotoxicity during Listeria infection.

关 键 词：神经前体细胞表达发育下调蛋白8 类泛素化蛋白酶8 T淋巴细胞 淋巴细胞活化 细胞分化 单核细胞增生李斯特菌 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]