Sertoli细胞对同种异体调节性T细胞/辅助性T细胞17分化平衡的调节作用

Modulation of Regulatory T Cell-Th17 Differentiation Balance by Allogeneic Sertoli

目的研究Sertoli细胞对同种异体免疫反应过程中调节性T细胞(Treg)/辅助性T细胞17(Th17)平衡的影响。方法体外分离并培养Sertoli细胞、T淋巴细胞和骨髓来源的DC细胞,将Sertoli、DC细胞分别与同种异体T细胞混合培养,光镜及电镜下观察Sertoli细胞形态,采用Western blot检测Sertoli细胞中人类主要组织相容性复合体(MHC)-Ⅱ、B7-H1蛋白的表达,收集培养上清液,采用酶联免疫吸附实验(ELISA)法检测Sertoli细胞培养上清内的转化生长因子(TGF)-β1、IL-17A等细胞因子水平;流式细胞术观察混合培养细胞分化结果。结果成功在体外培养Sertoli细胞;Western blot结果显示,Sertoli细胞与同种异体Nave CD4+T细胞(CD4+CD62L+CD25-)在体外混合培养时,其表面的MHC-Ⅱ类分子和B7-H1分子的表达量增加;ELISA结果显示,与同种异体Nave CD4+T细胞混合培养72h后,培养上清液中的TGF-β1浓度较单纯Sertoli培养组显著升高[(823.10±59.07)pg/mL vs.(174.89±23.51)pg/mL,P<0.01];流式细胞术结果显示,与同种异体Sertoli细胞共同培养的体系内,Nave CD4+T细胞(CD4+CD62L+CD25-)被诱导分化为CD25+Foxp3+Treg细胞的比例显著增加,从<1.0%增长至27.6%;用Sertoli细胞与Treg细胞混合培养上清液孵育DC细胞/Nave CD4+T细胞,可明显减少培养液中IL-17A的浓度,使其从(361.58±80.27)pg/mL降低至(137.54±52.19)pg/mL(P<0.05);同时IL-17A+T细胞亚群百分比也显著降低(27.2%降至17.9%)。结论Sertoli细胞体外培养产生的抗炎细胞因子和免疫抑制细胞因子能诱导Nave CD4+T细胞分化为CD25+Foxp3+Treg细胞,Sertoli细胞可能通过调节同种异体Treg/Th17分化平衡,诱导免疫耐受。

Objective To investigate the regulation of Sertoli cells on differentiation balance of allogeneic regulatory T cell （Treg）and T helper cell（Th17）.Methods Sertoli cells ,bone marrow‐derived dendritic cells（DC） and T lymphocytes were iso‐latedandculturedinvitro.SertolicellsandDCcellswereco‐culturedwithallogeneicTcells,respectively.MorphologyofSertoli cells were verified under the light microscope and electron microscope. The expression of major histocompatibility complex （MHC）‐Ⅱ and B7‐H1 in Sertoli cells was detected by Western blot.Concentration of cytokines[transforming growth factor （TGF）‐β1 and IL‐17A etc. ] in supernatant was determined by enzyme linked immunosorbent assay（ELISA）. The frequency of Treg cells differentiated from na？ve T cells was estimated by flow cytometry.Results Sertoli cells were successfully isolated and cultured in vitro.Western blot results showed the expression of MHC‐Ⅱ and B7‐H1 on Sertoli cells surface was increased significantly ,when Sertoli cells were co‐cultured with allogeneic na？ve CD4＋ T cells（CD4＋ CD62 L＋CD25）in vitro.ELISA re‐sults showed the TGF‐β1 concentration in culture supernatant was significantly increased [from （174.89 ± 23.51） pg/mL to （823.10 ± 59.07） pg/mL ,P〈0.05] .Flow cytometry results showed Na？ve T cells（CD4＋CD62 L＋CD25-）were induced to dif‐ferentiate into CD25＋ Foxp3＋ T cells（from 〈1.0% up to 27.6% ）by co‐cultured with Sertoli cells.And incubation with Sertoli/Treg supernatant（STS）significantly reduced the concentration of IL‐17A in the mixed culture supernatant of DC cells /Na？ve CD4＋ T cell[from （361.58 ± 80.27） pg/mL to （137.54 ± 52.19） pg/mL ,P〈0.05]. The percentage of IL‐17A＋ T cells subset was also significantly decreased（27.2% to 17.9% ,P〈0.05）.Conclusion The anti‐inflammatory cytokines and immune sup‐ 〈br〉 pressive cytokines derived from Sertoli cells can induce allogeneic na？ve T cells to differentiate into CD25＋ Foxp3＋ Treg cells in vitro.Sertoli cells can regulate the differentiation balance of allogeneic Treg/Th17 to induce the immune tolerance.