背景 研究表明,位于角膜中央区的树突状细胞(DCs)完全处于未成熟状态,而位于角膜周边区的DCs则大多处于成熟状态.角膜内的DCs广泛参与多种角膜相关疾病以及角膜移植免疫排斥反应,研究角膜内DCs的成熟状态具有重要意义.目的 探讨小鼠角膜基质细胞(CSCs)是否通过分泌转化生长因子β2(TGF-β2)以及前列腺素E2( PGE2)抑制DCs的成熟.方法 获取DCs、T细胞以及CSCs培养上清液.通过酶联免疫吸附实验( ELISA)测定CSCs培养上清液以及新鲜RPM1 1640培养基内PGE2和TGF-β2的质量浓度.在DCs成熟过程中,应用TGF-β2中和抗体以及PGE2受体阻滞剂AH6809,并按照处理方式的不同分为对照组、CSCs培养上清液组、AH6809组、TGF-β2中和抗体组、AH6809 +TGF-β2中和抗体组.采用流式细胞技术检测DCs细胞表型CD11c、CD80、CD86和MHC-Ⅱ的表达情况,通过葡聚糖内吞实验检测抗原吞噬功能,并通过混合淋巴细胞反应检测刺激T细胞增生的能力.结果ELISA检测结果显示,与新鲜RPMI 1640培养基相比,CSCs培养上清液内含有较高质量浓度的TGF-β2和PGE2.与CSCs培养上清液组比较,TGF-β2中和抗体组DCs CD80、CD86和MHC-Ⅱ的表达均升高,差异均有统计学意义(P＜0.05),葡聚糖的表达降低(P＜0.05),刺激指数(SI)增大(P＜0.05);AH6809组CD86和MHC-Ⅱ的表达均升高,葡聚糖的表达降低,SI增大,差异均有统计学意义(P＜0.05)；TGF-β2中和抗体+AH6809组DCs MHC-Ⅱ的表达和SI提高,差异均有统计学意义(P＜0.05).与对照组比较,TGF-β2中和抗体+AH6809组DCs CD80、CD86的表达和SI均较低,差异均有统计学意义(P＜0.05).结论体外培养的小鼠CSCs可以通过分泌TGF-β2及PGE2抑制DCs成熟,且这两种细胞因子可发挥叠加效应.%Background Researches demonstrated that dendritic cells(DCs) are uniformly immature in the central cornea but mature in the peripheral region of cornea.So an important question is which factor impact the maturation of DCs,especially in terms of corneal transplant rejection and the known roles of DCs in the development and persistence of some corneal diseases.Objective This study aimed to examine whether corneal stroma cells (CSCs) inhibit DCs maturation through secreting transforming growth factor beta 2 (TGF-β2) and prostaglandin E2 (PGE2).Methods DCs,T cells and CSCs were isolated and cultured from clean BALB/c and C57BL/6 mice.The level of PGE2 and TGF-β2in CSCs culture supernatant and the fresh RPMI 1640 medium were then analyzed by enzyme linked immunosorbent assay (ELISA).During the DCs maturation stage,the neutralizing TGF-β2 antibody and the EP2 receptor antagonist AH6809 were added in the CSCs culture supernatant respectively.According to the different treatment,cultured cells were assigned to different groups as follows:control group,CSCs culture supernatant group,AH6809 group,TGF-β2 antibody group,AH6809 +TGF-β2 antibody group.Subsequently,the cellular surface markers for DCs,including CD11c,CD80,CD86,and MHC- Ⅱ,were analyzed by flow cytometry.The capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions,and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran(FITC) uptake.Results The data of ELISA showed a higher concentration of TGF-β2 and PGE2 in murine CSCs culture supernatant than in the fresh RPMI 1640 medium.Compared with the CSCs culture supernatant group,the expression of CD80,CD86,and MHC- Ⅱ was up-regulated ( P ＜ 0.05 ),the expression of dextran was down-regulated ( P ＜ 0.05 ),and the stimulate index was increased( P＜ 0.05 ) in the TGF-β2 antibody group; the expression of CD86,and MHC-Ⅱ was up-regulated (P＜0.05),the expression of dextran was down-regulated ( F =13.740,P =0.006 ),and the stimulate index was increased(P＜0.05) in the AH6809 group;the expression of MHC-Ⅱ was up-regulated and the stimulate index was increased with statistical difference in interaction(P＜0.05 ) in the AH6809+TGF-β2 antibody group.Compared with the control group,the expression of CD80 and CD86,and the stimulate index was still lower(P＜0.05 ).Conclusions TGF-β2 and PGE2 contribute to the inhibitory effects on DCs maturation mediated by murine CSCs in vitro and further have additive effect on the immunosuppression of DCs.
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