臭氧暴露对哮喘大鼠CD4＋CD25highFoxp3＋调节性T淋巴细胞数量和Foxp3mRNA表达的影响

Effects of ozone exposure on percentage of CD4＋CD25highFoxp3＋ regulatory T cells and mRNA expression of Foxp3 in asthmatic rats

目的 探讨低浓度臭氧暴露对支气哮喘大鼠CD4＋CD25highFoxp3＋调节性T细胞数量及转录因子Foxp3mRNA表达的影响.方法 将60只雄性Wistar大鼠随机分为正常对照组、臭氧暴露组、哮喘组和臭氧暴露哮喘组,每组15只.哮喘组采用卵清白蛋白（OVA）致敏并吸入激发制备哮喘模型,正常对照组雾化生理盐水,臭氧暴露组予吸入低浓度臭氧,臭氧暴露哮喘组于每日雾化激发OVA前给予低浓度臭氧吸入.流式细胞仪检测外周血CD4＋CD25highFoxp3＋调节性T细胞占CD4＋T细胞的比例,酶联免疫吸附试验（ELISA）检测外周血和肺组织γ-干扰素（γ-INF）以及白细胞介素-4（IL-4）含量;聚合酶链反应（PCR）检测肺组织Foxp3mRNA表达水平.结果 哮喘组大鼠CD4＋CD25highFoxp3＋调节性T细胞占CD4T细胞的比例为6.12％±1.03％,臭氧暴露组为5.87％±1.26％,均低于正常对照组（9.85％±1.34％）;臭氧暴露哮喘组CD4＋CD25highFoxp3＋调节性T细胞占CD4T细胞的比例为（3.31％±0.85％）,低于正常对照组和哮喘组,差异均有统计学意义（P＜0.01）.哮喘组大鼠血浆和肺组织中IL-4含量[血浆：（21.83±5.12） ng/L,肺组织：（0.89±0.13） ng/L]高于正常对照组[血浆：（10.58±2.73） ng/L）,肺组织：（0.32±0.11） ng/L],差异有统计学意义（P＜0.01）;臭氧暴露哮喘组血浆和肺组织中IL-4含量[血浆：（35.47±7.24） ng/L,肺组织：（1.58±0.43） ng/L]高于正常对照组和哮喘组,差异有统计学意义（P＜0.01）.哮喘组血浆和肺组织中γ-INF含量[血浆：（61.78±23.45） ng/L,肺组织：（0.69±0.21） ng/L]低于正常对照组[血浆：（158.89±60.23） ng/L,肺组织：（1.86±0.29） ng/L],差异有统计学意义（P＜0.01）,臭氧暴露哮喘组γ-INF含量[血浆：（10.28±2.63） ng/L,肺组织：（0.46±0.12） ng/L低于正常对照组和哮喘组,差异有统计学意义（P＜0.01）.臭氧暴露哮喘组和哮喘组Foxp3mRNA表达低于正常对照组,差异均有统计学意义（P＜0.05）.结论 低浓度臭氧暴露可能通过下调CD4＋CD25highFoxp3＋调节性T细胞数量并抑制Foxp3mRNA表达,加重哮喘大鼠体内Th1/Th2比例失衡,提示臭氧暴露可能是诱发哮喘发病的因素之一.

Objective To investigate the effects of low-concentration ozone exposure on the percentage of CD4＋CD25highFoxp3＋ regulatory T cells and the mRNA expression of transcription factor Foxp3 in asthmatic rats.Methods Sixty male Wistar rats were randomly divided into 4 groups （n=15 for each）：normal control group,ovalbumin （OVA） exposure group,ozone exposure group,and OVA＋ozone exposure group.The OVA exposure group was sensitized and challenged with OVA to establish an asthma model; the normal control group inhaled aerosolized saline; the ozone exposure group inhaled low-concentration ozone; the OVA＋ozone exposure group inhaled low-concentration ozone before being challenged with aerosolized OVA every day.The percentage of CD4＋CD25highFoxp3＋ regulatory T cells in CD4＋ T cells was determined by flow cytometry.The levels of interferon-γ（INF-γ） and interleukin 4 （IL-4） in peripheral blood and lung tissue were measured by enzyme-linked im-munosorbent assay.The mRNA expression of Foxp3 in lung tissue was measured by PCR.Results The percentages of CD4＋CD25highFoxp3＋ regulatory T cells in OVA exposure group （6.12±1.03％） and ozone exposure group （5.87±1.26％） were significantly lower than that in normal control group （9.85±1.34％）,and the percentage of CD4＋CD25highFoxp3＋ regulatory T cells in OVA＋ozone exposure group （3.31±0.85％） was significantly lower than those in normal control group and OVA exposure group （P＜0.01）.The levels of IL-4 in plasma and lung tissue in OVA exposure group （plasma：21.83±5.12 ng/L; lung tissue：0.89±0.13 ng/L） were significantly higher than those in normal control group （plasma：10.58±2.73 ng/L; lung tissue：0.32±0.11 ng/L） （P＜0.01）.The levels of IL-4 in plasma and lung tissue in OVA＋ozone exposure group （plasma：35.47±7.24 ng/L; lung tissue：1.50±0.42 ng/L） were significantly higher than those in normal control group and OVA exposure group （P＜0.01）.The levels of INF-γin plasma and lung tissue in OVA exposure group （plasma：61.78±23.45 ng/L; lung tissue：0.69±0.21 ng/L] were significantly lower than those in normal control group [plasma：158.89±60.23 ng/L;lung tissue：1.86±0.29） （P＜0.01）.The levels of INF-γin plasma and lung tissue in OVA＋ozone exposure group （plasma：10.28±2.63 ng/L; lung tissue：0.41 ±0.12 ng/L） were significantly lower than those in normal control group and OVA exposure group （P＜0.01）.The mRNA expression of Foxp3 was significantly lower in the OVA＋ozone exposure group than in the normal control group （P＜0.05）.Conclusion Low-concentration ozone exposure may decrease the number of CD4＋CD25highFoxp3＋ regulatory T cells and inhibit the mRNA expression of Foxp3 to promote Th1/Th2 imbalance in asthmatic rats,suggesting that ozone exposure may be one of factors that induce asthma attack.