Galectin-9对活化的CD4+T细胞免疫调节作用的机制研究.doc

  Galectin-9 对活化的 CD4+T 细胞免疫调节 作用的机制研究# 栾弘，张茜，王艻，张妙，陈艳，徐小利，李杏爱，刘丽丽，袁劲，吕永曼** 5 10 15 20 25 30 35 40 （华中科技大学同济医学院附属同济医院肾内科，武汉 430030） + + anti-CD3(2.5ug/ml)抗体、anti-CD28(5ug/ml)抗体和 IL-2(100ng/ml)刺激，培养 3 天。将 + + + 和 Th17 细胞的比例，同时利用 Annexin-检测各组细胞早期凋亡情况；并利用 Elisa 检测 淋巴细胞分泌 IFN-γ、IL-4、IL-10、IL-12、IL-17A 和 TGF-β1 等细胞因子的水平；利用 Western Blot 检测 T-bet、Gata-3 和 ROR-γt 等 T 细胞分化调控蛋白的变化。结果 与正 常对照组和 Galectin-9+α-lactose 组相比，Galectin-9 组于 2h 时开始出现细胞形态改 + + 减少（P＜0.05），但 Th2 细胞无明显变化；培养上清液中 IFN-γ、IL-12、IL-17A 和 TGF- β1 的表达水平降低（P＜0.05）,而 IL-4 和 IL-10 等 Th2 型细胞因子水平无明显变化；同 时 T-bet 和 ROR-γt 的表达水平减少（P＜0.05）。结论 Galectin-9 抑制活化 CD4+T 细胞 的 Th1 和 Th17 型免疫反应，而对 Th2 型免疫反应无影响,免疫调节的机制可能与其在转录水 平影响 Th1 和 Th17 特定转录因子的表达有关。 关键词：Galectin-9；Tim-3；Th1；Th2；Th17 中图分类号：R392 The mechanism of Galectin-9 in the immune regulation of the active CD4+T cells LUAN Hong, ZHANG Qian, WANG Le, ZHANG Miao, CHEN Yan, XU Xiaoli, LI Xingai, LIU Lili, YUAN Jin, LV Yongman (Department of Nephrology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology, WuHan 430030) Abstract: Objective To study the immune regulation of Galectin-9 on the active CD4+T cells and demonstrate the mechanisms. Methods Lymphocytes were harvested from wild-type C57BL/6 mouse, from which na?ve CD4+T cells were separated via MACS and then stimulated with anti-CD3 Ab(2.5ug/ml),anti-CD28 Ab(5ug/ml) and IL-2(100ng/ml) for 3 days. The active CD4+T cells were divided into 3 groups: Control group, Galectin-9 group and Galectin-9+α-lactose group. We detected the cell proliferation level by CFSE fluorescence intensity and then dynamically observed the cell morphological changes. The proportion of CD4+CD69+T, Th1, Th2 and Th17 cell was valued and the early apoptosis of each group was also detected by Annexin-Ⅴ-PI method； Meanwhile, Elisa was used to detect the cytokine levels of IFN-γ, IL-4, IL-10, IL-12, IL-17A and TGF-β1 secreted by lymphocytes；Also Western Blot was used to observe the changes of T cell differentiation r