Ag85 B 调节小鼠髓样树突状细胞的成熟和TSLP 介导下 TSLPR 和 OX40 L 的表达

摘要

AIM:To investigate the maturation of mice immature myeloid dendritic cells (mDCs) induced by antigen(Ag)85B of mycobacterium tuberculosis, and the expression of TSLPR and OX40L mediated by TSLP in vitro. METHODS:Recombinant mouse GM-CSF ( rmGM-CSF) and rmIL-4 were used to induce bone marrow precursor cells of C57BL/6 mice to differentiate into immature mDCs in vitro.mDCs were identified followed by purification using CD 11c binding magnetic beads .The morphological characteristic of mDCs was observed under inverted phase-contrast microscope and scanning electron microscope .The surface phenotypes of mDCs were determined by flow cytometry .To obtain the opti-mal concentrations of Ag85B and TSLP, immature mDCs were cultured with different concentrations of Ag 85B or TSLP at 0 (control group), 50, 100 and 200 μg/L for 24 h, and the expression of cell surface molecules CD 80, CD86, TSLPR and OX40L was detected by flow cytometry.In addition, the expression of TSLPR and OX40L in Ag85B and TSLP-co-stimula-ted mDCs was determined by flow cytometry .RESULTS:After 7 d of culture in vitro, the cells showed irregular dendritic protrusions under the inverted-phase contrast microscope , and had wrinkles and dendritic splits under scanning electron mi-croscope , conformed to the morphological characteristics of immature mDCs .The mDCs cells expressed higher level of spe-cific marker CD11c, lower level of co-stimulatory molecules CD80 and CD86, which conformed to the phenotype of imma-ture mDCs.The CD80 +and CD86 +cell ratios of mDCs displayed significant increases in 50, 100 and 200μg/L Ag85B or TSLP groups compared with control group (P<0.05).The ratios of TSLPR +and OX40L+cells did not differ among dif-ferent concentrations of Ag 85B groups.The ratios of TSLPR +and OX40L+cells were significantly increased in 100 μg/L and 200μg/L TSLP groups compared with control group and 50μg/L TSLP group (P<0.05).Under the circumstance of optimal Ag85B or TSLP treatment concentration at 200 μg/L, there was significantly decreased in TSLPR and OX 40L cell ratio of mDCs in Ag85B group or Ag85B combined with TSLP group when compared with TSLP group (P<0.05), and no significant difference among Ag85B group, Ag85B combined with TSLP group and control group was observed .CONCLU-SION: Ag85B enhances mDCs maturation by up-regulating the expression of co-stimulatory molecules CD80 and CD86, and inhibit the expression of pro-inflammatory specific molecules TSLPR and OX40L on TSLP-activated mDCs, indicating that Ag85B modifies the development of asthmatic airway inflammation through the pathway of TSLP -activated mDCs.%目的：研究抗原85B（ Ag85B）体外诱导小鼠未成熟的髓样树突状细胞（ mDCs）的成熟以及对胸腺基质淋巴细胞生成素（TSLP）介导下mDCs表达TSLP受体（TSLPR）和OX40L的影响，探究Ag85B抑制哮喘气道炎症的可能机制。方法：应用重组小鼠GM－CSF和IL－4体外诱生C57BL／6小鼠未成熟的mDCs，并运用免疫磁珠分离的方法纯化，采用光镜和扫描电镜、流式细胞术进行形态学观察和细胞表型鉴定；分别用0、50、100、200μg／L不同浓度的Ag85B或TSLP作用于纯化并鉴定后的mDCs，培养24 h，流式细胞术检测细胞表面分子CD80、CD86、TSL－PR和OX40L的表达，选取最佳的Ag85B或TSLP处理浓度。随后将 mDCs随机分为空白对照组、Ag85B处理组、TSLP处理组和Ag85B＋TSLP处理组，培养24 h后检测mDCs的促炎表面分子TSLPR和OX40L的表达。结果：体外诱导培养7 d，倒置相差显微镜下可见细胞表面呈现不规则树突样突起，扫描电镜下见细胞类圆形，表面有少量皱褶和较少分叉的树突状突起，符合未成熟mDCs的形态学特点；纯化后的mDCs表达表面分子CD11c的细胞较表达共刺激分子CD80和CD86的细胞多，符合未成熟mDCs 的表型特征。与空白对照组比较，50～200μg／L的Ag85B处理组mDCs表达CD80和CD86的细胞比率显著增高（P＜0．05），表达TSLPR和OX40L的细胞比率无显著差异。与空白对照组相比较，50、100和200μg／L浓度的TSLP处理组的mDCs表达CD80和CD86的细胞比率均显著增加（ P＜0．05）；与空白对照组和50μg／L TSLP处理组相比较，100μg／L和200μg／L TSLP处理组的mDCs表达TSLPR和OX40L的细胞比率均显著升高（ P＜0．05）。选取200μg／L作为Ag85B和TSLP的优化作用浓度，结果发现Ag85B处理组和Ag85B＋TSLP处理组的mDCs表达TSLPR和OX40L的细胞比率较TSLP处理组均显著降低（ P＜0．05），与空白对照组比较差异不显著。结论：Ag85B可通过上调mDCs 表达共刺激分子CD80和CD86促进其成熟，同时下调TSLP介导的mDCs表达促炎表面分子TSLPR和OX40L，推测Ag85B可能通过TSLP介导的mDCs途径抑制气道炎症。