咪喹莫特对致敏大鼠支气管旁淋巴结T辅助淋巴细胞亚群产生细胞因子的影响

Modulation of T-lymphocyte subsets production with imiquimod in the PBLN cell cultures from ovalbumin-sensitized rats

目的研究免疫调节剂咪喹莫特对卵蛋白(OVA)致敏大鼠支气管旁淋巴结(PBLN)细胞培养体系中T辅助淋巴细胞(Th)亚群产生细胞因子的影响,并探讨其作用机制。方法建立PBLN细胞培养体系,并按不同浓度分为A～F组进行干预。在培养0、3、6、12、24、48h后,各组分别取5孔细胞培养液,用酶联免疫吸附法(ELISA)检测各细胞因子的蛋白质水平,用逆转录聚合酶链反应(RTPCR)检测细胞沉淀中白细胞介素(IL)4和干扰素(IFN)γ等细胞因子的mRNA表达。结果在A组的各时间点PBLN细胞培养液中,仅可检测到少量IFNγ。随着培养时间的延长,咪喹莫特浓度为1、10μg/ml的E和F组与B组相比,IL4及相关mRNA水平增加缓慢,IFNγ水平增长迅速(均P<0.005)。此作用从培养6h开始,12h达峰值,持续至24h。在培养12～48h时,C组IL4表达与B组比,差异有统计学意义(P<0.05),而IFNγ表达无差异(P>0.05)。结论咪喹莫特对致敏大鼠PBLN细胞培养体系中Th亚群产生细胞因子的最佳作用发生在12h时,提示咪喹莫特可能在支气管哮喘等特异性疾病的迟发炎症反应阶段发挥重要作用。

Objective To observe the effect and mechanism of imiquimod on T helper （Th） cell subsets in the Parabronchial lymph node （PBLN） cell cultures from ovalbumin （OVA）-sensitized rats. Methods PBLN were isolated and cultured. PBLN cells were divided into A ～ F, according to different concentrations of intervention. Cultured for 0, 3, 6, 12, 24, 48 hours, the expressions of IL-4 and IFN-γ in supernatants were determined by ELISA. The mRNA expressions of the cytokines in cells weredetected by RT-PCR. Results In the group A, only low concentrations of IFN-γ were detected. Based on the cultured time, the concentrations of IFN-γ were increased significantly if imiquimod concentration was between 1 and 10 μg/ml. Levels of IL-4 were increased slowly compared with those in the group B （P 〈0. 005）. The effect began at 6h, achieved peak at 12 h, continued 24 h. Between 12 h and 48 h, significant differences of IL-4 levels were found in the group C. The levels of IFN-γ in the group C were found no difference compared with those in group B （ P 〉 0. 05 ）. Conclusion Imiquimod show the best effect on antigen-specific Th cell subsets when cultured for 12h. The results suggest that imiquimod have benefit in atopic diseases such as the late inflammation reaction of asthma.