肽聚糖对树突细胞分泌促炎性细胞因子以及实验性自身免疫性葡萄膜炎辅助性T细胞17的影响  

The effect of peptidoglycan on the secretion of pro-inflammatory cytokines by dendritic cells and the regulation of T helper 17 responses in experimental autoimmune uveitis

作　　者：肖青[1] 粘红[1] 魏瑞华[1] 张晓敏[1] 李雪[1] 刘勋[1] 李筱荣[1] 

机构地区：[1]天津医科大学眼科医院眼科研究所,300384

出　　处：《中华眼底病杂志》2013年第6期600-604,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：基金项目：天津市自然科学基金（13JCYBJC23300）

摘　　要：目的观察肽聚糖（PGN）对树突细胞（DCs）分泌促炎性细胞因子的影响以及对实验性自身免疫性葡萄膜炎（EAU）辅助性T细胞（Th）17（Thl7细胞）反应的调控。方法利用粒细胞-巨噬细胞集落刺激因子、白细胞介素（IL）-4（IL-4）定向诱导健康小鼠骨髓细胞向DCs分化。诱导分化的第6天将DCs分为对照组与实验组，实验组加入PGN干预，对照组加入等量无菌磷酸缓冲盐溶液，培养24h后利用实时荧光逆转录聚合酶链反应（RT—PCR）技术，检测2组培养细胞中Th17细胞分化相关的细胞因子IL-23、肿瘤坏死因子-α（TNF-α）、IL-6、IL-18mRNA相对表达量。采用光感受器间维生素A类结合蛋白（IRBP）1—20多肽片段（IRBP1—20）和弗氏不完全佐剂及结核分枝杆菌H37RA免疫C57BL／6小鼠。免疫后13d分离EAU模型鼠脾脏和淋巴结的IRBP1—20特异的T细胞与经PGN干预或未干预的DCs共培养。收集共培养后2d的细胞，实时荧光RT—PCR检测维甲酸相关孤儿受体7t（RORyt）、IL-17、T—bet、7干扰素（IFN-γ）mRNA相对表达量；此外，收集共培养后2、5、7d的细胞分别进行流式细胞仪分析，观察Th17、Th1细胞的变化。结果实时荧光RT—PCR检测结果显示，实验组DCs经PGN干预后IL-23、IL-1G、IL-6、TNF—αmRNA相对表达量较对照组显著升高，差异有统计学意义（t=-14．363、-5．627、-3．85、-28．151；P〈0．05）；实验组RORγt、IL-17mRNA相对表达量较对照组显著升高（t=-5．601、-19．76；P〈0．05），而Tbet、IFN7mRNA相对表达量较对照组显著降低（f-4．717、11．207；P〈0．05）。流式细胞仪检测结果显示，与对照组相比，实验组的DCs与T细胞共培养后2、5、7d，IL-17＋细胞的百分比升高（t=-2．944、-3．03、-4．81；P〈0．05），而IFN-γ细胞的比例变化不大（t=-1.25、-0．18、-2．16；P〉0．05）。结论PGN能够刺激DCs分泌IL-23、TNF—α、IL-6及IObjective To investigate the effect of peptidoglycan （PGN） on the secretion of pro- inflammatory cytokines by dendritic cells （DCs） and the regulation of T helper 17 （Th17） responses in experimental autoimmune uveitis. Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin （IL）-4 to induce DCs. DCs cultured for six days were randomly divided into two groups： PGN-treated group and control group. The DCs in PGN- treated group were stimulated with PGN and the same volume of phosphate buffered saline was added to the DCs as control group. The relative mRNA expression levels of IL-23, tumor necrotic factor α （TNF-α）, IL 6, IL-1βwere measured by real-time reverse transcriptase polymerase chain reaction （RT-PCR）. Peptide fragment of interphotoreceptor retinoid binding protein （IRBP1 20）-specific T cells, which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 clays earlier, were co-cultured with PGN-treated or untreated DCs, respectively. Total RNA from T cellscocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor α （ROR-α）, IL-17, T-box expression in T cells （T-bet）, interferon γ （IFN γ） mRNA were detected by real-time RT-PCR. On the second, the fifth and the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-7, IL-17 positive cells. Results The real-time RT-PCR results revealed that the level of IL-23, IL-113, IL-6, TNF-γγ mRNA from PGN-stimulated DCs were significantly increased compared to the control group （t= - 14. 363, - 5. 627, - 3.85, - 28. 151; P〈0.05）. The level of RORγt, IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group （t=-5. 601, -19.76; P〈0.05）. However, the level of T-bet, IFN-γ mRNA from the T cells cocultured with PGN-stimulated

关 键 词：肽聚糖 药理学 葡萄膜炎 病理生理学 TOLL样受体2 T细胞抗原受体特异性 Th17细胞 

分 类 号：R773[医药卫生—眼科]