终止香烟烟雾暴露后大鼠Th1/Tc1介导气道炎症及调节性T细胞的变化

Change of airway inflammation induced by Th1/Tc1 and the expression of T regulatory cells in smoking cessation rats

目的 观察香烟烟雾暴露和终止香烟烟雾暴露后大鼠Th1/Tc1介导气道炎症及支气管肺泡灌洗液（BALF）中调节性T细胞（Treg）的变化.方法 将50只健康清洁级雄性Wistar大鼠随机分为5组：12周正常对照组（简称12周对照组）、24周正常对照组（简称24周对照组）、12周香烟烟雾暴露组（简称12周暴露组）、24周香烟烟雾暴露组（简称24周暴露组）、终止香烟烟雾暴露组（简称终止暴露组）.烟熏法复制大鼠气道炎症的动物模型.12周后,12周对照组、12周暴露组处置取材,24周暴露组继续烟熏12周,终止暴露组终止烟雾暴露12周,将后2组及24周对照组处置取材.HE染色观察小气道病理改变,进行气道评分 收集BALF进行细胞学计数和分类计数 酶联免疫吸附法（ELISA）法测BALF中4种细胞因子：Th1/Th2型细胞因子干扰素（IFN）γ、白细胞介素（IL）4及促炎因子IL-8、肿瘤坏死因子（TNF）α的浓度 流式细胞术检测各组大鼠BALF中Treg细胞的比例,RT-PCR检测各组大鼠BALF中Foxp3 mRNA的表达.结果 （1）12周暴露组、24周暴露组、终止暴露组气道炎症评分较12周对照组、24周对照组明显高（均P＜0.01）.24周暴露组、终止暴露组气道炎症评分均较12周暴露组高（均P＜0.01）.（2）与12周对照组、24周对照组相比,12周暴露组、24周暴露组、终止暴露组BALF中IFN-γ、TNF-α和IL-8高,IL-4低（均P＜0.01）.与12周暴露组相比,终止暴露组IFN-γ、IL-4、TNF-α差异均无统计学意义（均P＞0.05）,IL-8较高（P＜0.01）,24周暴露组IFN-γ、TNF-α和IL-8明显高（均P＜0.01）.（3）BALF中Treg细胞比例,12周暴露组（7.4%±0.8%）、24周暴露组（7.8%±1.7%）、终止暴露组（7.0%±1.4%）较12周对照组（4.8%±1.2%）、24周对照组（4.7%±1.2%）高（均P＜0.01）,前3组之间BALF中Treg细胞比例差异均无统计学意义（均P＞0.05）.（4）12周暴露组（0.22±0.02）、24周暴露组（0.23±0.03）、终止暴露组（0.20±0.04）BALF中Foxp3 mRNA表达较12周暴露组（0.13±0.01）、24周暴露组（0.11±0.02）高（均P＜0.01）.前3者之间BALF中Foxp3 mRNA表达差异均无统计学意义（均P＞0.05）.结论 香烟暴露致大鼠气道Th1/Tc1介导炎症伴Treg细胞表达增高,终止香烟烟雾暴露后其炎症及Treg细胞高表达仍持续存在,提示该免疫失衡可能是导致终止香烟暴露后Th1/Tc1气道炎症仍持续进展的原因之一.

Objective To study the change of airway inflammation induced by Th1/Tc1 and the expression of CD4 ＋ CD25 ＋ regulatory T cells （Treg） in smoking cessation rats. Methods Fifty healthy male Wistar rats were randomly divided into five groups： 12-week normal control （group A, n = 10）, 24-week normal control （group B, n = 10）, 12-week smoke exposure （group C, n = 10）, 24-week smoke exposure （group D, n = 10） and smoking cessation （group E, n = 10）. Groups C, D and E were exposed to cigarettes for 12 weeks. At Week 12, groups A and C were sacrificed. Group D continued smoke exposure and group E had smoking cessation for 12 weeks. At Week 24, groups B, D and E were sacrificed.Pathomorphological changes of small airway were analyzed. The cells in BALF （bronchoalveolar lavage fluid） were collected and analyzed by absolute and differential cell counts. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of IFN-γ, IL-4, IL-8 and TNF-α And flow cytometry was employed to determine the Foxp3 ＋ Treg cell populations and reverse transcription-polymerase chain reaction （RT-PCR） to assay the mRNA expression for Foxp3. Results （ 1 ） Compared with groups A and B, the airway inflammation score of groups C, D and E increased significantly （ all P 〈 0.01 ）. Compared with group C, the airway inflammation score of groups D and E both increased （all P 〈 0.01 ）, especially group D （2） compared with groups A and B, the levels of IFN-γ, TNF-α and IL-8 in groups C, D and E increased （all P 〈0.01 ） while those of IL-4 decreased. The levels of IFN-γ, TNF-α and IL-8 showed no difference between groups C and E. The levels of IFN-γ, TNF-α and IL-8 were higher in group D than those in groups C and E （3） the ratio of Foxp3 ＋ Treg cells in BALF was higher in group C（7.4% ±0.8% ）,group D （ 7.8% ± 1.7%）and group E（7.0% ± 1.4%）than group A（4.8% ± 1.2%）and group B （4.7% ± 1.2% ） （ all P 〈0.01 ）. There were no differences in the ratio of Foxp3 ＋ Treg cells among groups C, D and E （all P 〈 0.05 ） （4） there was an elevated expression of Foxp3 mRNA in group C （0.22 ±0.02）, group D（0.23 ±0.03）, group E（0.20 ±0.04） versus group A（0.13 ±0.01 ）and group B（0.11 ±0.02）（ all P 〈 0.01 ）. But there was no difference in the expression of Foxp3 mRNA among groups C, D and E （all P 〉 0.05 ）. Conclusions Airway inflammation induced by Th1/Tc1 and an elevated expression of Treg cells in BALF are found in smoke exposure rats. Upon smoking cessation, the above-mentioned airway inflammation still persists and the expression of Treg cells in BALF shows no decrease. It suggests that an immune imbalance may be involved in the progression of Th1/Tc1-induced airway inflammation upon smoking cessation.