短期与长期烟草烟雾暴露小鼠中CD4＋Foxp3＋调节性T细胞的变化及意义

摘要/Abstract

摘要： 目的: 观察短期与长期烟草烟雾暴露小鼠肺组织和外周血CD₄^＋Foxp3^＋调节性T细胞(Treg细胞)及相关细胞因子的变化, 探讨Treg细胞在烟草诱导气道慢性炎症发生中的作用。方法: 将60只健康清洁级雄性BALB/c小鼠随机分为长期暴露24周组、短期暴露4周组和对照组, 每组20只。用烟草烟雾暴露法建立小鼠气道炎症模型。收集支气管肺泡灌洗液(BALF)进行细胞学计数和分类;用流式细胞术检测Treg/CD₄^＋T细胞比例;用实时荧光定量PCR法检测肺组织Foxp3 mRNA的表达;采用ELISA检测小鼠血清和BALF中IL-6、TGF-β和IL-10水平。结果: 短期暴露组肺组织和外周血中Treg细胞比例分别为(5.24±0.86)%和(5.24±1.23)%, 均显著高于对照组[(2.52±0.62)%和 (3.54±0.87)%];而长期暴露组肺组织和外周血中Treg细胞比例分别为(1.83±0.39)％和(1.88±0.25)％, 均显著低于对照组, 差异均有统计学意义(均P<0.01)。短期暴露组肺组织Foxp3 mRNA表达量为 2.67±0.73, 显著高于对照组(1.49±0.37);而长期暴露组Foxp3 mRNA表达量为 0.61±0.21, 显著低于对照组(1.49±0.37)(均P<0.01)。长期暴露组外周血IL-6和TGF-β分别为(56.47±19.41) pg/mL、 (144.22±43.19) ng/mL, 均显著高于短期暴露组[ (6.22±2.06) pg/mL、 (23.32±8.32) ng/mL]和对照组[ (5.12±1.48) pg/mL、(18.14±13.00) ng/mL], 差异均有统计学意义(均P<0.01)。长期暴露组外周血IL-10浓度为(4.04±2.57) pg/mL, 显著低于对照组的(8.26±2.02) pg/mL;而短期暴露组外周血IL-10浓度为(10.42±2.45) pg/mL, 显著高于对照组(P<0.01)。结论: 短期与长期烟草暴露可导致小鼠Treg细胞比例和相关细胞因子改变, 提示Treg细胞可能参与气道炎症发生的免疫调节。对烟草暴露引起Treg失衡的研究为气道炎症相关的COPD的防治提供新的思路及策略。

关键词: 调节性T细胞, 烟草暴露, 气道炎症, 慢性阻塞性肺疾病, 模型, 动物

Abstract: AIM : To investigate the changes of Treg and related cytokines in cigarette smoke-induced mice model of chronic airway inflammation, and to explore the role of Treg in the pathogenesis of COPD.METHODS: Sixty male BALB/c mice were randomly divided into 3 groups: a 24 wk smoke chronic exposure group, a 4 wk sub-acute exposure group and control group(n=20 each). Cells in BALF were collected and analyzed by absolute and differential cell counts. IL-6, TGF-β and IL-10 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD₄^＋Foxp3^＋Treg in peripheral blood mononuclear cell (PBMC) and lung tissue were determined by flow cytometry. The mRNA expressions of Foxp3 in lung tissues were measured by real-time PCR.RESULTS:Ratio of Treg was higher in the sub-acute smoke-exposure group in lung tissue(5.24％±0.86%)and peripheral blood (5.24％±1.23%) compared with the control group[(2.52±0.62)%, (3.54±0.87)%]. But the ratio of Treg in the chronic exposure group in lung tissue (1.83％±0.39％) and peripheral blood (1.88％±0.25％)was lower compared with the control group, all P<0.01. The level of Foxp3 mRNA in lung tissue was increased in the sub-acute smoke-exposure (2.67±0.73) compared with the control groups (1.49±0.37).But it was remarkably decreased in the chronic exposure group (0.61±0.21) compared with the control groups, P<0.01. Levels of IL-6 and TGF-β were remarkably increased in the chronic exposure group in serum[(56.47±19.41) pg/mL, (144.22±43.19 pg/mL)] compared with the sub-acute smoke-exposure group[(6.22±2.06 pg/mL), (23.32±8.32 pg/mL)] and the control group[(5.12±1.48 pg/mL), (18.14±13.00 pg/mL)], all P<0.01. IL-10 in serum was significantly decreased in the chronic smoke-exposure group (4.04±2.57 pg/mL) compared with the control group(8.26±2.02 pg/mL), and it was increased in sub-acute smoke-exposure group (10.42±2.45 pg/mL) compared with the control group, all P<0.01.CONCLUSION: Our study thus reveals that the change of Treg and the related cytokines exists in mice with sub-acute and chronic cigarette smoke exposure, suggesting its potential role in the breakdown of immune self-tolerance in chronic airway inflammatory response. Further research on Treg change in airway inflammation of COPD may provide insights into the development of new therapeutic targets for this disease.

Key words: regulatory T cells, cigarette exposure, airway inflammation, chronic obstructive pulmonary disease, model, animals

中图分类号:

R965.2

俞万钧, 王华英, 翁跃颂, 宋嫣瑾, 顾霄, 应华娟. 短期与长期烟草烟雾暴露小鼠中CD₄^＋Foxp3^＋调节性T细胞的变化及意义[J]. 中国临床药理学与治疗学, 2014, 19(6): 601-606.

YU Wan-jun, WANG Hua-ying, WENG Yue-song, SONG Yan-jin, GU Xiao, YING Hua-juan. Variation of CD₄^＋Foxp3^＋ regulatory T cells in sub-acute and chronic cigarette smoke exposure in mice[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(6): 601-606.

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短期与长期烟草烟雾暴露小鼠中CD₄^＋Foxp3^＋调节性T细胞的变化及意义

Variation of CD₄^＋Foxp3^＋ regulatory T cells in sub-acute and chronic cigarette smoke exposure in mice

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