RAGE在高迁移率族蛋白B1调控CD4＋T细胞Th1／Th2功能性分化中的作用

The role of RAGE in high mobility group box-1 protein1-mediated CD4＋T cells differentiation to Th1/Th2

目的 探讨晚期糖基化终产物受体（receptor for advanced glycation endproducts, RAGE）在高迁移率族蛋白B1（high mobility group box-1 protein, HMGB1）介导CD4＋T细胞Th1/Th2功能性分化中的作用。方法 体外分离、培养小鼠脾脏CD4＋T 淋巴细胞，将细胞浓度调整为2×10^6/L，接种至96 孔培养板，0.2 mL/ 孔，预先加入终浓度为3 μg/L的刀豆素A（ConA）刺激12 h；依据HMGB1刺激剂量随机分成4组：对照组、10 ng/mL组、100 ng/mL组和1 000 ng/mL，分别在HMGB1刺激12 h、24 h和48 h 时间点，利用酶联免疫吸附试验（enzyme-linked immunosorbentassay，ELISA）法检测白介素-4（IL-4）和干扰素-γ（IFN-γ）表达；根据实验结果，在后期实验中选择100 ng/mL HMGB1刺激时间为24 h；将ConA刺激后的细胞细胞随机分成4组：对照组、A组（HMGB1刺激）、B组（HMGB1＋PBS刺激）、C组（HMGB1＋RAGE抗体刺激）。A、B、C组分别加入PBS 稀释的终浓度为100 ng/L HMGB1工作液，对照组加入等容积的PBS液；其中B、C组在HMGB1刺激之前加入PBS 液或PBS 液1：200 稀释的终浓度为5 μg/L RAGE中和抗体孵育2 h；分别采用Western-blot和荧光定量PCR 法测定RAGE、CATA-3的表达；ELISA 法检测白介素-4（IL-4）和干扰素-γ（IFN-γ）表达，多组之间的数据比较采用单因素方差分析，组间两两比较采用LSD-t 检验，以P〈0.05 为差异有统计学意义。结果与对照组（6.07±0.25）比较，100ng/mL HMGB1刺激24 h（4.43±0.17）、48 h（3.67±0.09），IFN-γ/IL-4 显著下降（P〈0.05）；而与对照组比较（6.34±0.42），10 ng/mL HMGB1刺激24 h（7.65±0.19），IFN-γ/IL-4比值显著增加（P〈0.05），而100 ng/mL（5.10±0.19）和1 000 ng/mL HMGB1（3.31±0.12）刺激24 h IFN-γ/IL-4 显著下降（P〈0.05）；与对照组比较，A组RAGE 蛋白和mRNA 表达显著升高（P〈0.05）；A、B组IFN-γ/IL-4比值（A组 5.75±0.27；B组 5.66±0.31）较对照组（7.37±0.36）明显降低（P〈0.05），GATA3 mRNA 显著升高（P〈0.05）；而C组IFN-γ/IL-4比值（6.19±0.14）较A组升高（P〈0.05），GATA3 mRNA 下降（P〈0.05），A组与B组比较差异无统计学意义（P〉0.05）；与A组比较，B组RAGE的表达差异无统计学意义（P〉0.05），C组明显降低（P〈0.05），而C组较对照组RAGE的表达仍有明显增加（P〈0.05）。结论 HMGB1体外介导的CD4＋T 淋巴细胞向Th2 功能性分化至少部分是通过过度活化RAGE/GATA3途径来实现的。

Objective To explore the role of receptor for advanced glycation end products （RAGE） in HMGBl-mediated CD4＋T cells differentiation to Thl/Th2. Methods CD4＋ T lymphocytes isolated from the spleens of male BALB/C mice by magnetic beads were suspended in RPMI-1640 with 10% FCS in 2×10^6cell/well on 96-well cell culture plates in vitro. The cells were randomly divided 4 groups according to concentration of HMGB1 treatment： control group, 10 ng/mL group, 100 ng/mL group, 1 000 ng/mL group after stimulation with ConA in 3μg/mL for 12 hours. IL-4 and IFN-3, levels in culture supernatants were quantitated by ELISA kits after HMGB1 stimulation for 12, 24, and 48 h. According to the results, cultured cells were exposed to HMGB1 in 100 ng/mL for 24 h in the following experiments. The cells were randomly divided into 4 groups： control group, A group, B group, C group, and each group were cultured with ConA in 3μg/mL for 24 h. The cells of control group and other three groups were stimulated with PBS or 100 μg/L HMGB1 for 24 h. The cells of B, C groups were incubated with 1/200 diluted 5μg/L anti-RAGE Abs （anti-bodies） or PBS for 2 h before HMGB1 stimulation. The cell suspension was obtained to detect the levels of IL-4 and IFN-γ by EILSA and the protein levels and mRNA expressions of RAGE, CATA-3 were detected by western-blot and real-time fluorescent quantitative PCR, respectively. Results Compared with control group, CD4＋T cells incubated with increasing concentrations of HMGB1 （100, 1 000 ng/mL） for 24 h resulted in a decrease in IFN-y/IL-4 ratio （P〈0.05）. When CD4＋T cells were exposed to 100 ng/mL HMGB1 for 12 h, IFN-γ/IL-4 ratio was markedly increased. However, CD4＋T cells treated with 100 ng/mL HMGB1 for 24, 48 h, IFN-γ/1L-4 ratio was markedly inhibited （P〈0.05）. Compared with control group, protein levels and mRNA expressions of RAGE and GATA3 of cells in A group were significantly increased （P〈0.05）, and IFN-γ/IL-4 ratio of cell suspension in A group and B group was significantly down-regulated （P〈0.05）. Compared with A group, IFN-γ/IL-4 ratio of cell suspension in C group was increased （P〈0.05）, and expression of GATA3 mRNA was down-regulated （P〈0.05）. Compared with A group, protein level of RAGE of cells in C group was significantly down-regulated （P〈0.05）, but protein level of RAGE of cells in C group was still increased compared with control group （P〈0.05）. Conclusion Th1/Th2 differentiation induced by HMGB1 on CD4＋T lymphocytes in vitro was at least partly mediated by over-activating RAGE/GATA3 pathway.