LCMV-CL13慢性感染小鼠模型的建立及其BCR突变分析  

Establishment of chronic infection mouse model by LCMV-CL13 virus and analysis of BCR mutation

作　　者：李哲 蔡方舟 李丹 陈倩 苑一真 王卫 LI Zhe;CAI Fangzhou;LI Dan;CHEN Qian;YUAN Yizhen;WANG Wei(Comparative Medicine Center,Peking Union Medical College(PUMC).Institute of Medical Laboratory Animal Science,Chinese Academy of Medical Sciences(CAMS).Beijing Key Laboratory for Animal Models of Emerging and Reemerging Infectious.NHC Key Laboratory of Human Disease Comparative Medicine,Beijing 100021,China)

机构地区：[1]北京协和医学院比较医学中心,中国医学科学院医学实验动物研究所,新发再发传染病动物模型研究北京市重点实验室,卫健委人类疾病比较医学重点实验室,北京100021

出　　处：《中国实验动物学报》2022年第2期177-184,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然基金面上项目(81873963);医科院创新工程项目(2021-I2M-1-035)。

摘　　要：目的建立淋巴细胞性脉络丛脑膜炎病毒LCMV-CL13慢性感染小鼠模型,分析其作为B细胞受体高频突变研究模型的可能性。方法C57BL/6N小鼠经尾静脉途径接种2×10^(6)PFU剂量LCMV-CL13病毒,感染后第10、20、30、40、50、60、70天采集样品,通过qPCR检测组织病毒载量,流式检测分析外周血CD4^(+)T、CD8^(+)T、CD19^(+)B细胞及脾生发中心B细胞比例,免疫组库测序分析BCR V区基因丰度及突变率。结果在LCMV-CL13病毒感染小鼠体内检测到1×10^(6)copies/μL水平的病毒复制;在感染平台期,小鼠外周血CD4^(+)T细胞比例逐渐升高(13.15%±0.72%),CD8^(+)T细胞先降低(2.17%±0.40%)后逐渐恢复(6.65%±0.52%),CD19^(+)B细胞以及生发中心B细胞比例分别增加至(40.32%±0.46%)和(10.03%±0.60%);测序结果证明BCR重链V基因使用频率下降、突变率显著性升高(P<0.05)。结论成功建立LCMV-CL13慢性感染小鼠模型;此模型可用于BCR突变研究,为研究慢性病毒感染导致的B细胞高频突变奠定了基础。Objective To establish an LCMV-CL13 chronic infection mouse model and analyze its use for B cell somatic hypermutation research.Methods C57BL/6N mice were inoculated with 2×10^(6) plaque-forming units of LCMV-CL13 virus via tail vein injection.The tissue viral load was then detected by quantitative polymerase chain reaction on days 10,20,30,40,50,60,and 70 post-infection.The CD4^(+)and CD8^(+)T cell and CD19^(+)B cell ratios in peripheral blood and the germinal center B cell percentage of the spleen were determined by flow cytometry,and the gene abundance and BCR V region mutation rate were analyzed using immune repertoire technology.Results LCMV-CL13-infected mice maintained a tissue viral load of 1×10^(6) copies/μL virus.The percentage of CD4^(+)T cells in the peripheral blood gradually increased in the infection plateau phase to(13.15%±0.72%),while the percentage of CD8^(+)T cells first decreased to(2.17%±0.40%)and then gradually recovered to(6.65%±0.52%),and the percentages of CD19^(+)B cells and germinal center B cells increased to(40.32%±0.46%)and(10.03%±0.60%),respectively.Sequencing result demonstrated that the frequency of V gene usage decreased and the mutation rate of the heavy chain CDR3 V gene increased 1.7-fold with increasing infection time.Conclusions We successfully established a LCMV-CL13 chronic infection mouse model.This model can be used to study BCR mutations,and provides a research tool for investigating B cell somatic hypermutation caused by chronic viral infection.

关 键 词：淋巴细胞性脉络丛脑膜炎病毒 高频突变 生发中心B细胞 B细胞受体 

分 类 号：Q95-33[生物学—动物学]