LAG3对多房棘球蚴感染小鼠模型CD8^(+)T细胞免疫功能调节作用的研究  

Effect of lymphocyte activation gene 3(LAG3)on immune function of CD8^(+)T cells in a mouse model of Echinococcus multilocularis infection

作　　者：阿比旦·艾尼瓦尔 孜比姑·肉素 阿迪莱·多力坤 邓冰清 李静[1,2] 王慧 张传山 Abidan Ainiwaer;Zibigu Rousu;Adilai Duolikun;DENG Bingqing;LI Jing;WANG Hui;ZHANG Chuanshan(College of Basic Medical,Xinjiang Medical University,Urumqi 830017,China;Clinical Medicine Research Institute,the First Afilicated Hospital of Xinjiang Medical University,Urumqi 830054,China;Xinjiang Uygur Autonomous Region,Urumqi 830000,China)

机构地区：[1]新疆医科大学基础医学院,乌鲁木齐830017 [2]新疆医科大学第一附属医院临床医学研究院,乌鲁木齐830054 [3]新疆维吾尔自治区地方病分子生物学重点实验室,乌鲁木齐830000

出　　处：《新疆医科大学学报》2024年第5期660-667,674,共9页Journal of Xinjiang Medical University

基　　金：国家杰出青年科学基金培育项目(xyd2021J003);新疆维吾尔自治区自然科学基金项目(2022D01D60,2022D01E51)。

摘　　要：目的研究淋巴细胞激活基因3(Lymphocyte activation gene 3,LAG3)对多房棘球蚴慢性感染小鼠CD8^(+)T细胞免疫功能的调节作用。方法取C57BL/6野生型(Wild-type,WT)和LAG3缺陷型(Knock-out,KO)小鼠各10只,每只小鼠经肝门静脉接种3000个多房棘球蚴原头节建立多房棘球蚴感染模型。感染12周后,分别取两组小鼠肝脏和脾脏组织,采用苏木精-伊红(Hematoxylin-eosin,HE)染色观察肝脏病灶周围炎性细胞浸润和病理表现,通过免疫组织化学观察肝脏病灶周围“炎症微环境”与脾脏中CD8^(+)T的比例。收集两组小鼠肝脏与脾脏淋巴细胞,采用流式细胞术筛选不同CD8^(+)T细胞亚群,检测CD8^(+)T细胞、效应记忆CD8^(+)T细胞(Effector memory CD8^(+)T cell,CD8^(+)Tem)、中心记忆CD8^(+)T细胞(Central memory CD8^(+)T cell,CD8^(+)Tcm)、初始CD8^(+)T细胞(Naive CD8^(+)T cell,CD8^(+)Tn)比例与绝对数,以及分泌细胞因子γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)、白细胞介素10(IL-10)和白细胞介素17A(IL-17A)的比例。通过多房棘球蚴虫体蛋白体外刺激两组小鼠肝脏淋巴细胞,采用流式细胞术检测CD8^(+)T细胞分泌细胞因子IFN-γ、IL-10和IL-17A的比例。结果HE染色结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏形成的炎性病灶数量增多,差异有统计学意义(P<0.05)。免疫组织化学结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏病灶周围CD8^(+)T细胞募集升高,差异有统计学意义(P<0.05);LAG3缺陷型小鼠脾脏CD8^(+)T细胞募集有降低的趋势,差异无统计学意义(P>0.05)。流式细胞术结果显示,与野生型小鼠比较,LAG3缺陷型小鼠肝脏效应记忆型CD8^(+)T细胞比例有升高的趋势,差异无统计学意义(P>0.05);脾脏CD8^(+)T细胞比例降低,脾脏CD8^(+)Tem表型比例升高,差异均有统计学意义(P<0.01)。LAG3缺陷型小鼠肝脏和脾脏CD8^(+)T细胞分泌TNF-α和IL-10能力均增强,差异均有统计学意义(P<0.05)。多房�Objective To investigate the regulatory role of lymphocyte activation gene 3(LAG3)on the immune function of CD8^(+)T cells in mice chronically infected with Echinococcus multilocularis(E.multilocularis).Methods 10 C57BL/6 wild-type(WT)and LAG3 knock-out(KO)mice were used,with each mouse receiving an intraportal injection of 3000 protoscoleces of E.multilocularis to establish an infection model.12 weeks post-infection,liver and spleen tissues were collected from both groups.Hematoxylin-eosin(HE)staining was performed to observe inflammatory cell infiltration and pathological changes around liver lesions,while immunohistochemistry was utilized to assess the ratio of CD8^(+)T cells in the“inflammatory microenvironment”surrounding liver lesions and within the spleen.Liver and spleen lymphocytes were isolated from both groups,and flow cytometry was employed to identify different CD8^(+)T cell subsets,measuring the proportions and absolute numbers of CD8^(+)T cells,effector memory CD8^(+)T cells(CD8^(+)Tem),central memory CD8^(+)T cells(CD8^(+)Tcm),and naive CD8^(+)T cells(CD8^(+)Tn),as well as their secretion of cytokines interferon-gamma(IFN-γ),tumor necrosis factor-alpha(TNF-α),interleukin-10(IL-10)and interleukin-17A(IL-17A).Additionally,liver lymphocytes from both groups were stimulated ex vivo with E.multilocularis antigen,and flow cytometry was used to assess the ratios of IFN-γ,IL-10 and IL-17A secreted by CD8^(+)T cells.Results HE staining showed that LAG3 KO mice had significantly more inflammatory foci in their livers compared to WT mice(P<0.05).Immunohistochemical analysis revealed that CD8^(+)T cell recruitment around liver lesions was significantly higher in LAG3 KO mice than in WT mice(P<0.05),while there was a non-significant trend toward reduced CD8^(+)T cell recruitment in the spleens of LAG3 KO mice(P>0.05).Flow cytometry results indicated that the proportion of effector memory CD8^(+)T cells in the livers of LAG3 KO mice showed a non-significant increasing trend compared to WT mice(P>0.05);ho

关 键 词：多房棘球蚴 多房棘球蚴病 淋巴细胞激活基因3 CD8^(+)T细胞 

分 类 号：R53[医药卫生—内科学]