解脲脲原体GrpE蛋白促进小鼠树突状细胞成熟并诱导Th1型免疫应答  

Ureaplasma urealyticum GrpE promotes mouse dendritic cell maturation and elicits Th1 immune responses

作　　者：郭方毅 唐艳红 袁红霞 张文君 向璟 刘鹏琴 滕文友 李冉辉 代国知 Guo Fangyi;Tang Yanhong;Yuan Hongxia;Zhang Wenjun;Xiang Jing;Liu Pengqin;Teng Wenyou;Li Ranhui;Dai Guozhi(Center of Laboratory Medicine,Chenzhou Hospital Affiliated to Hengyang Medical School of University of South China,the First People′s Hospital of Chenzhou,Chenzhou 423000,China;Pathogenic Biology Institute of Hengyang Medical School of University of South China,Hengyang 421000,China;State Key Laboratory of Respiratory Diseases,Guangzhou Medical University,Guangzhou 510180,China)

机构地区：[1]南华大学衡阳医学院附属郴州医院/郴州市第一人民医院检验医学中心,郴州423000 [2]南华大学衡阳医学院病原生物学研究所,衡阳421000 [3]广州医科大学呼吸疾病国家重点实验室,广州510000 [4]怀化市第一人民医院核医学科,怀化418000

出　　处：《中华微生物学和免疫学杂志》2022年第1期41-49,共9页Chinese Journal of Microbiology and Immunology

基　　金：湖南省研究生科研创新项目(CX20200982);湖南省卫生健康委科研计划课题项目(B2019003);湖南省科技创新计划项目(2018SK50302);湖南省教育厅项目(19B496);湖南省自然科学基金(2020JJ4521)。

摘　　要：目的:探讨解脲脲原体GrpE蛋白(Ureaplasma urealyticum GrpE,Uu-GrpE)对树突状细胞成熟的影响及其对T细胞极化的作用。方法:表达纯化Uu-GrpE蛋白并利用Western blot鉴定。分离培养小鼠骨髓来源树突状细胞(bone marrow-derived dendritic cells,BMDCs),利用LDH试剂盒分析Uu-GrpE的细胞毒性,流式细胞术检测Uu-GrpE对BMDCs表面共刺激分子CD80和CD86以及主要组织相容性复合体Ⅱ类分子(major histocompatibility complexⅡ,MHCⅡ)表达的影响,ELISA检测其刺激BMDCs后IL-12p70、TNF-α、IL-1β和IL-6等细胞因子的表达;磁珠分选同系异体小鼠的脾脏中初始CD4^(+)T细胞(CD4^(+)Naive T细胞),通过构建BMDCs与Naive T细胞共培养体系分析Uu-GrpE刺激成熟的BMDCs(GrpE-BMDCs)对Naive T细胞增殖及极化的影响。GrpE-BMDCs经尾静脉注射免疫小鼠并利用ELISA和流式细胞术观察其所诱导的特异性体液和细胞免疫应答。结果:成功表达Uu-GrpE并分离出高纯度BMDCs,Uu-GrpE可刺激BMDCs分泌IL-12p70、TNF-α、IL-1β和IL-6等细胞因子且无细胞毒性;同时Uu-GrpE可显著增加BMDCs表面分子CD80[平均荧光强度(mean flourscence indensity,MFI):(324.00±22.11)vs(91.03±10.95),P<0.01]、CD86[MFI:(1176.00±51.39)vs(217.00±14.93),P<0.01]和MHCⅡ[MFI:(708.70±56.32)vs(185.70±16.77),P<0.01]的表达;与单独GrpE-BMDCs组和GrpE(热变性)-BMDCs^(+)T细胞组比较,GrpE-BMDCs不仅可刺激Naive T细胞增殖[刺激指数(stimulation index):(7.25±0.21),(6.55±0.23),(6.09±0.35),P均<0.05],并且还诱导Naive T细胞分泌Th1型细胞因子IL-2和IFN-γ[IL-2:(145.60±14.67)pg/ml,(55.92±3.12)pg/ml,(26.05±2.40)pg/ml,P<0.05和P<0.01;IFN-γ:(267.20±37.80)pg/ml,(146.70±20.65)pg/ml,(27.84±6.69)pg/ml,P均<0.05],而Th2型细胞因子IL-4、IL-10和IL-5则无明显改变。与PBS组和PBS^(+)BMDCs组比较,GrpE-BMDCs经过继转移后,可诱导机体产生Th1型免疫应答。结论:Uu-GrpE蛋白明显促进BMDCs成熟及活化,且进一步证明其作为解脲脲原体候选蛋Objective To investigate the effects of Ureaplasma urealyticum GrpE(Uu-GrpE)on the maturation of dendritic cells and the polarization of T cells.Methods Uu-GrpE was expressed and purified,and then identified by Western blot.The cytotoxicity of Uu-GrpE to mouse bone marrow-derived dendritic cells(BMDCs)was analyzed by LDH kit.After stimulating BMDCs with Uu-GrpE,the expression of costimulatory molecules,CD80,CD86 and major histocompatibility complexⅡ(MHCⅡ),on the surface of BMDCs was detected by flow cytometry,and ELISA was used to detect the cytokines such as IL-12p70,TNF-α,IL-1βand IL-6.CD4^(+)Naïve T cells were isolated from mouse spleen tissues by magnetic beads.A co-culture system of BMDCs and Naïve T cells was constructed to analyze the effects of GrpE-stimulated mature BMDCs(GrpE-BMDCs)on T cell proliferation and polarization towards Th1/Th2.Mice were immunized with GrpE-BMDCs through the tail vein,and the induced humoral and cellular immune responses were detected by ELISA and flow cytometry.Results Uu-GrpE was successfully express and high purity BMDCs were isolated.Uu-GrpE could stimulate BMDCs to secrete cytokines such as IL-12p70,TNF-α,IL-1βand IL-6 without having cytotoxicity.Uu-GrpE significantly increased the expression of CD80[mean flourscence indensity(MFI):(324.00±22.11)vs(91.03±10.95),P<0.01],CD86[MFI:(1176.00±51.39)vs(217.00±14.93),P<0.01]and MHCⅡ[MFI:(708.70±56.32)vs(185.70±16.77),P<0.01]on BMDCs.Compared to the GrpE-BMDCs only group and GrpE(boiled)-BMDCs^(+)T cell group,the GrpE-BMDCs^(+)T cell group showed significantly increased T cell proliferation[stimulation index:(7.25±0.21)vs(6.55±0.23)and(6.09±0.35),both P<0.05],and dramatically promoted T cell secretion of IL-2 and IFN-γ[IL-2:(145.60±14.67)pg/ml vs(55.92±3.12)pg/ml and(26.05±2.40)pg/ml,P<0.05 and P<0.01;IFN-γ:(267.20±37.80)pg/ml vs(146.70±20.65)pg/ml and(27.84±6.69)pg/ml,both P<0.05].However,no significant change was observed in the expression of Th2-type cytokines.Moreover,the adoptive transfer of GrpE-

关 键 词：解脲脲原体 GRPE 树突状细胞 免疫应答 

分 类 号：R392[医药卫生—免疫学]