摘要目的 观察香烟暴露及断烟后大鼠Treg细胞的变化,探讨Treg细胞与断烟后大鼠肺部炎症反应和肺气肿持续存在的关系.方法 将50只雄性Wistar大鼠按随机数字表法分为5组:健康对照组、香烟暴露组和断烟组,其中健康对照组和香烟暴露组又分为12周组(对照1组和实验1组)和24周组(对照2组和实验2组).香烟烟雾暴露法建立大鼠肺气肿模型.HE染色观察大鼠肺气肿的改变,酶联免疫吸附法检测大鼠BALF中白细胞介素(IL-8)和肿瘤坏死因子-α(TNF-α)水平,流式细胞术检测大鼠外周血和肺实质中CD4+Foxp3调节性T细胞(Treg细胞)比例,实时定量PCR法检测大鼠外周血和肺实质中Foxp3的mRNA表达.多组间比较采用单因素方差分析,组内两两比较采用SNK和Games-Howell检验.结果 实验1组和实验2组平均内衬间隔(MLI)为(64.9±5.3)μm和(77.9±11.5)μm,均明显高于对照1组和对照2组的(39.0±3.8)μm和(40.3±2.7)μm,差异均有统计学意义(P＜0.01);断烟组MLI[(71.5 ±5.8)μm]介于实验1组和实验2组之间,但肺气肿程度较实验1组明显加重,差异有统计学意义(P＜0.01).实验1组和实验2组IL-8水平[(68±17)ng/L和(85±16)ng/L]及TNF-α水平[(14.1±1.8)ng/L和(20.1±8.7)ng/L]均明显高于对照1组和对照2组[(44±8)ng/L、(43±9)ng/L及(6.3±2.3)ng/L、(5.8±1.6)ng/L],差异均有统计学意义(P＜0.05).实验1组和实验2组肺实质中CD+4Foxp3+Treg细胞[(6.6±0.8)%和(5.3±0.9)%]明显少于对照1组和对照2组[(9.0±1.0)%和(9.6±0.9)%],差异均有统计学意义(P＜0.01);断烟组肺实质中CD+4Foxp3+Treg细胞[(7.2±0.6)%]与实验1组比较虽无明显减少,但未能恢复至正常水平.实验1组和实验2组肺实质中Foxp3的mRNA表达量(17±7和9±7)明显低于对照1组和对照2组(39±6和42±7),差异均有统计学意义(P＜0.01);断烟组肺实质中Foxp3的mRNA表达量(21±9)与实验1组比较未见明显减少,但未能恢复至正常水平.结论 香烟暴露肺气肿大鼠肺内调节性T细胞下调可能导致大鼠肺内炎症反应放大,并在断烟后持续存在,提示调节性T细胞下调可能是大鼠肺气肿持续进展的原因之一.

abstractsObjective To evaluate the expression of Treg in a cigarette smoke-induced rat model of emphysema and after smoking cessation in the rats. Methods Fifty male Wistar rats were randomly divided into control group 1 (12 weeks), control group 2 (24 weeks), smoke-exposure group 1 (12 weeks),smoke-exposure group 2 (24 weeks) and smoking cessation group, with 10 rats in each group. Alveolar airspace enlargement was observed by hematoxylin-eosin (HE) staining. IL-8 and TNF-α levels in bronchoalveolar lavage fluid (BALF) were tested by ELISA. The proportion of CD4+ Foxp3+ Treg in peripheral blood and lungs of rats was determined by flow cytometry. The mRNA expression of Foxp3 was measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test was used for comparison between 2 groups. Results The mean linear intercept (MLI) in smoke-exposure group 1 and group 2 [( 64. 9 ± 5. 3 ) μm, (77.9 ± 11.5 ) μm] was higher than those in the control group 1 and group 2 [( 39. 0 ± 3. 8 ) μm, (40. 3 ± 2. 7 ) μm], all P ＜ 0. 01.Compared with smoke-exposure group 2, the MLI in smoking cessation group (71.5 ± 5. 8) μm showed a lower value ( P ＜ 0. 01 ), but still higher than that in smoke-exposure group 1 ( P ＜ 0. 01 ). The IL-8 and TNF-α levels in BALF of smoke-exposure group 1 and group 2 [(68 ± 17) ng/L, (85 ± 16) ng/L],[( 14. 1 ± 1.8)ng/L, (20. 1 ±8. 7) ng/L] were higher than those in control group 1 and group 2 [(44 ±8)ng/L, (43±9)ng/L], [(6.3 ±2.3) ng/L, (5.8±1.6)ng/L], all P＜0.05. The IL-8 and TNF-αlevels were not statistically different between in smoking cessation group (56±6)ng/L, (14.7±4.7)ng/L and smoke-exposure group 1. The percentage of Treg in the lungs of smoke-exposure group 1 and group 2 [(6. 6 ±0. 8)%, (5.3 ±0. 9)%] was significantly decreased as compared to control group 1 and group 2 [(9.0 ± 1.0 ) %, ( 9. 6 ± 0. 9 ) %], all P ＜ 0. 01. The percentage of Treg in lungs was not statistically different between smoke-exposure group 1 and smoking cessation group (7. 2 ±0. 6)%. In peripheral blood,there was no significant difference between groups in the percentage of Treg. In the lung, Foxp3 mRNA expression in smoke-exposure group 1 and group 2 [( 17 ±7), (9 ±7)] was less than that in control group 1 and group 2 [( 39 ± 6 ), (42 ± 7 )], all P ＜ 0. 01. The Foxp3 mRNA expression was not statistically different between smoke-exposure group 1 and smoking cessation group (21 ±9). No significant differences in peripheral blood Foxp3 mRNA expression was found between groups. Conclusions Decreased Treg was present in lungs of cigarette smoke-induced model of emphysema despite 12 weeks' smoking cessation,suggesting that down-regulation of Treg may be involved in the amplified and persistent inflammation after smoking cessation.