Notch1信号通路调控银屑病模型小鼠Th17细胞分化和功能

Notch1 signaling pathway regulates Th17 cell differentiation and function in murine psoriasis model

目的:探讨Notch1信号通路对银屑病模型小鼠Th17细胞分化和功能的调控作用。方法:以5%咪喹莫特外涂联合α-2b干扰素腹腔注射的方法制备20只银屑病模型小鼠,免疫磁珠分离小鼠脾脏CD4^+T淋巴细胞,流式细胞术检测Th17细胞比例,实时荧光定量RT-PCR检测Th17细胞特异性转录因子RORγt、效应性细胞因子IL-17A、Notch1信号分子及其靶基因Hes-1的mRNA表达水平,并与10只对照组小鼠相比较。将银屑病模型小鼠CD4^+T淋巴细胞分为未干预对照组和Notch1抑制剂组(γ-分泌酶抑制剂DAPT),检测DAPT阻断Notch1信号对银屑病模型小鼠Notch1信号分子及Hes-1、Th17细胞比例、RORγt及IL-17A表达水平的影响。结果:银屑病模型小鼠CD4^+T淋巴细胞中Th17细胞比例,RORγt、IL-17A、Notch1及Hes-1的mRNA表达水平均显著高于对照小鼠[分别为(2.97±0.86)%比(0.65±0.11)%,t=15.083;(5.75±0.61)比(1.57±0.43),t=21.630;(7.83±0.97)比(1.63±0.31),t=25.348;(7.10±1.37)比(1.47±0.34),t=17.386;(7.30±1.15)比(1.67±0.48),t=18.840,P均<0.01];与未干预对照组相比,银屑病模型小鼠CD4^+T淋巴细胞各DAPT处理组中Notch1、Hes-1mRNA表达水平,Th17细胞比例、RORγt与IL-17A mRNA表达水平及培养上清液中IL-17A含量均明显下降,组间比较差异具有统计学意义(F值分别为74.368、89.719、126.572、94.558、124.323和123.231,P均<0.01),且随DAPT浓度的增加呈剂量依赖性降低。结论:Notch1信号通路能够调控银屑病模型小鼠Th17细胞的分化和功能,对银屑病的免疫靶向治疗有潜在价值。

Objective：To determine the effect of Notchl signaling pathway on the differentiation and function of Thl7 cells in murine psoriasis model. Methods： BALB/c mice were randomly divided into psoriasis model group and control group. Murine psoriasis model was established by topical 5% imiquimod application in combination with intraperitoneal injection of α-2b interferon. The CD4＋ T lymphocytes were isolated by magnetic activated cell sorter （MACS）. Flow cytometric analysis （FCM） was performed to detect the per- centage of Thl7 cells. Real-time RT-PCR was employed to measure the mRNA levels of ROR＇yt, IL-17A, Notchl and Hes-1. The CD4＋ T lymphocytes were then divided into γ-secretase inhibitor DAPT groups and control group, and the expression differences of Notchl signaling molecule and its target gene Hes-1 mRNA levels,Thl7 cell percentage, RORγt and IL-17A mRNA levels, and IL-17A concen- trations in cell-free supernatant were detected. Results： The expression levels of Thl7 cell percentage and RORγt,IL-17A,Notchl and Hes-1 mRNA in CD4＋ T lymphocytes of murine psoriasis model were significantly higher than control mouse [ （2. 97 ±0. 86 ）% vs. （0.65±0. 11）%,t=15.083;（5.75±0.61） vs. （1.57±0.43）,t=21.630;（7.83±0.97） vs. （1. 63±0.31）,t=25.348;（7.10± 1.37） vs. （ 1.47±0. 34）, t = 17. 386; （7. 30 ± 1.15 ） vs. （ 1.67±0. 48 ）, t = 18. 840, respectively, all P〈0. 01 ]. Compared with control group, Thl7 cell percentage, mRNA expression levels of Notchl, Hes-1, RORγ/t and IL-17A, and IL-17A concentrations in cell-free su- pematant from cultured CD4＋ T lymphocytes of murine psoriasis model were dramatically decreased in DAPT treated groups in a dose- dependent way （ F = 74. 368,89. 719,126. 572, 94. 558,124. 323 and 123.231 respectively, all P 〈 0. 01 ）. Condusion： Notchl signaling pathway can regulate the differentiation and function of Thl7 cells in murine psoriasis model, and may have potential value for the target immunotherapy of psoriasis.