海藻酸钠/乙二醇壳聚糖双网络水凝胶通过诱导自噬增强CD4+CD25+调节性T细胞免疫抑制活性的研究OA
Enhancement of CD4+CD25+regulatory T cell immunosuppressive activity via autophagy induction by sodium alginate/glycol chitosan double-network hydrogels
目的 研究海藻酸钠/乙二醇壳聚糖(Alg/GC)双网络水凝胶对CD4+CD25+调节性T细胞(Tregs)功能的调控作用及其影响Tregs功能的关键细胞内机制.方法 制备Alg/GC双网络水凝胶,将Tregs暴露于磷酸盐缓冲液(PBS,PBS对照组)、Alg/GC双网络水凝胶(Alg/GC组)、雷帕霉素(雷帕霉素组)、Alg/GC+3-甲基腺嘌呤(Alg/GC+3-MA组)中24 h.流式细胞术检测叉头翼状螺旋转录因子、细胞毒性T淋巴细胞抗原4(CTLA-4)的表达水平和CD4+CD25-效应T细胞的增殖活性;采用酶联免疫吸附试验检测细胞因子水平,包括γ干扰素(IFN-γ)、白介素(IL)-10、IL-2、IL-4和转化生长因子β(TGF-β);采用蛋白质印迹法、透射电子显微镜及激光共聚焦显微镜观察自噬相关蛋白的表达水平和自噬小体的形成.结果 经Alg/GC双网络水凝胶处理后,Alg/GC组和雷帕霉素组LC3-Ⅱ、Beclin1表达水平显著高于PBS对照组,P62蛋白表达水平显著低于PBS对照组(P<0.05);利用激光共聚焦显微镜检测内源性LC3-Ⅱ在Tregs中的分布,Alg/GC组和雷帕霉素组LC3斑点数量显著高于PBS对照组(P<0.05);透射电子显微镜可见Tregs中出现明显的自噬小体.在Tregs免疫抑制功能检测实验中,Tregs细胞CTLA-4表达及TGF-β分泌水平在Alg/GC+3-MA组及Beclin1敲除组中分别较对照组均显著下降(P<0.05),Tregs对CD4+CD25-效应T细胞增殖活性抑制作用较相应对照组均显著减弱(P<0.05),共培养后CD4+CD25-效应T细胞分泌IL-4/IFN-γ比值较对照组均显著下降(P<0.05).结论 Alg/GC双网络水凝胶可显著激活Tregs自噬活性.该自噬活性的上调可能是介导Alg/GC双网络水凝胶增强Tregs免疫抑制功能的核心机制之一.
Objective To investigate the regulatory effects of sodium alginate/glycol chitosan(Alg/GC)double-network hydrogels on the function of CD4+CD25+regulatory T cells(Tregs)and to elucidate the key intracellular mechanisms underlying these effects.Methods Sodium alginate/glycol chitosan(Alg/GC)double-network hydrogels were prepared.Tregs were exposed to phosphate buffered saline(PBS,PBS group),Alg/GC double-network hydrogels(Alg/GC group),rapamycin(rapamycin group)and Alg/GC+3-methyladenine(3-MA,Alg/GC+3-MA group)for 24 hours.Expressions of forkhead wing helix transcription factor and cytotoxic T lymphocyte antigen 4(CTLA-4),as well as the proliferation activity of CD4+CD25-effector T cells were measured by flow cytometry.Enzyme linked immunosorbent assay(ELISA)was used to determine the cytokine levels including interferon-γ(IFN)-γ,interleukin(IL)-10,IL-2,IL-4,and transforming growth factor β(TGF)-β.The expression of autophagy-associated proteins and formation of autophagosomes were evaluated by Western blot,transmission electron microscope and laser scanning confocal microscopy,respectively.Results After treatment with the Alg/GC double-network hydrogels,the expression levels of LC3-Ⅱ and Beclin1 in the Alg/GC group and the Rapamycin group were significantly higher than those in the PBS control group,while the expression level of P62 was significantly lower than that in the PBS control group(P<0.05).The distribution of endogenous LC3-Ⅱ in Tregs was detected by laser scanning confocal microscope,and the number of LC3 puncta in the Alg/GC group and the Rapamycin group was significantly higher than that in the PBS control group(P<0.05).Transmission electron microscope observed obvious autophagosomes in Tregs.In the experiment assessing the immunosuppressive function of Tregs,the expression of CTLA-4 and the secretion level of TGF-β in Tregs were significantly decreased in the Alg/GC+3-MA group and the Beclin1 knockout group compared with the control group,respectively(P<0.05).The inhibitory effect of Tregs on the proliferation activity of CD4+CD25-effector T cells was significantly weakened compared with the control group(P<0.05).Furthermore,the ratio of IL-4/IFN-γ secreted by CD4+CD25-effector T cells after co-culture was significantly reduced compared with the control group(P<0.05).Conclusions The Alg/GC double-network hydrogels can significantly activate the autophagic activity of Tregs.The upregulation of autophagic activity may be one of the core mechanisms mediating the enhancement of the immunosuppressive function of Tregs by Alg/GC double-network hydrogels.
陈坤威;潘申奥;潘丽娜;姚咏明
温州医科大学附属第一医院急诊科,浙江 温州 325000解放军总医院医学创新研究部,北京 100853温州医科大学附属第一医院麻醉科,浙江 温州 325000解放军总医院医学创新研究部,北京 100853
调节性T细胞海藻酸钠乙二醇壳聚糖自噬免疫调控
regulatory T cellssodium alginateglycol chitosanautophagyimmune regulation
《感染、炎症、修复》 2026 (2)
143-151,9
国家重点研发计划项目(2202YFA1104600)温州市科技局项目(Y20210710)
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