【作者】 唐海娟；

【导师】 钟小宁；

【作者基本信息】 广西医科大学 ， 呼吸内科学， 2012， 硕士

【摘要】 目的观察烟草烟雾暴露及断烟后小鼠肺部CD4+IL-17+辅助性T细胞(Th17)数量及活性表达的变化,探讨Thl7细胞与断烟后小鼠肺部炎症和肺气肿的关系。方法50只雄性Balb/c小鼠,分为5组(按照随机数字表法)：每组10只,对照12周组(C1组)、对照24周组(C2组)、烟草烟雾暴露12周组(S1组)、烟草烟雾暴露24周组(S2组)、断烟组(烟草烟雾暴露12周后断烟12周组)(S3组)。按照烟草烟雾暴露法,建立小鼠肺气肿及断烟模型。经HE染色后,观察小鼠肺气肿病理的改变,计算肺平均内衬间隔(Lm)、肺泡破坏指数(DI)；酶联免疫吸附(ELISA)法检测小鼠肺实质中肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)水平；免疫荧光定量PCR法检测小鼠肺实质中维甲酸相关孤独受体(RORyt)和白细胞介素-17(IL-17)的mRNA表达；流式细胞术检测小鼠肺实质中CD4+IL-17+T(Th17)细胞占CD4+T细胞的百分比,并分析这些指标的相互关系。结果S1组和S2组的Lm[(39.19±3.51)μm和(46.87±7.16)μm]及DI[(39.13±1.57)和(45.16±3.13)]均明显高于Cl组和C2组的Lm[(32.38±3.73)μm和(32.60±3.21)μm]及DI[(28.23±1.62)和(28.86±2.07)],以S2组增高更为明显,与S2组相比,S3组的Lm[(43.16±1.73)μm]及DI[(43.01±2.04)]有所减轻,但与S1组比较,肺气肿程度仍然继续加重,差异均有统计学意义(P<0.05)。在肺组织匀浆上清液中S1组和S2组TNF-α及IL-6水平[(77.244±13.68)ng/L和(101.71±18.98)ng/L]及[(558.5±68.6)ng/L和(669.1±99.7)ng/L]均明显高于C1组和C2组[(62.10±16.11)ng/L和(64.37±15.12)ng/L]及[(492.5±28.1)ng/L和(484.0±28.1)ng/L],以S2组增高更为明显,与S2组相比,S3组TNF-α及IL-6水平[(87.36±8.37)ng/L]及[(612.4±22.0)ng/L]有所降低,但与S1组比较,S3组TNF-α及IL-6水平仍然明显增高,差异均有统计学意义(P<0.05)。S1组和S2组肺实质中RORyt mRNA及IL-17mRNA表达量：[(25.50±3.75)和(43.10±5.76)]及[(25.50±3.03)和(43.40±5.38)]较C1组和C2组：[(10.00±4.83)和(11.20±7.35)]及[(12.70±5.12)和(8.30±6.07)]明显增高,以S2组增高更明显,与S2组相比,S3组RORyt mRNA及IL-17mRNA表达量[(37.70±5.36)]及[(37.60±5.13)]有所减少,但与S1组比较,S3组RORyt mRNA及IL-17mRNA表达量仍然明显增高,差异均有统计学意义(P<0.05)。S1组、S2组肺实质中Th17细胞的比例[(3.27±1.12)%、(7.19±2.24)%]明显高于C1组、C2组[(1.80±0.75)%、(1.99±0.59)%],以S2组增高更为明显,与S2组相比,S3组肺实质中Th17细胞比例[(4.57±1.15)%]有所降低,但与S1组比较,S3组肺实质中Th17细胞比例仍然明显增高,差异均有统计学意义(P<0.05)；S1、S2及S3组小鼠的肺实质中Th17细胞比例、RORyt mRNA和IL-17mRNA表达量与平均内衬间隔(Lm)及肺泡破坏指数(DI)均呈显著正相关(r值分别为0.709及0.674、0.802及0.812和0.705及0.766,均P<0.05)。结论Th17细胞在烟草烟雾暴露小鼠肺内表达增高,即使断烟12周后,Th17细胞仍然上调,未能恢复正常水平,提示Th17细胞上调有可能是导致烟草烟雾暴露肺气肿发生并在断烟后炎症持续不退的原因之一。更多还原

【Abstract】 Objective To evaluate the expression of Th17cells in lungs of cigarette smoke-induced mice and smoking cessation mice,and to explore the relationship between Th17cells and lung inflammation or emphysema of smoking cessation mice.Methods There are Fifty male BALB/c mice, which were randomly divided into5groups, with10mice in every group, including a smoke-exposure12weeks group (S1), a smoke-exposure24weeks group (S2), a smoking cessation group (smoke-exposure12weeks and then with12weeks’smoking cessation)(S3), a control group12weeks (C1), a control group24weeks (C2). Morphological changes were evaluated by Lm (mean linear intercepts) and DI (destructive index). ELISA(Enzyme linked immunosorbent assay) was used to detect the levels of TNF-a and IL-6. The mRNA expression of IL-17and RORyt was measured by real-time PCR. The proportion of CD4+IL-17+T cell in lungs of mice was determined by flow cytometry.Results Mean linear intercepts in S1and S2groups [(39.19±3.51)μm and (46.87±7.16)μm]and DI in S1and S2groups[(39.13±1.57) and (45.16±3.13)] were significantly higher than those in C1and C2groups [(32.38±3.73)μm and (32.60±3.21)μm] and [(28.23±1.62) and (28.86±2.07)]. Compared with S2group, the Lm and DI in S3group [(43.16±1.73)μm] and [(43.01±2.04)] showed a lower value, but still higher than those in S1group, all P<0.05. The levels of TNF-a in S1and S2groups [(77.24±13.68) ng/L and (101.71±18.98)ng/L] and IL-6in S1and S2groups [(558.5±68.6) ng/L and (669.1±99.7) ng/L] were higher than those in C1and C2groups[(62.10±16.11) ng/L and (64.37±15.12) ng/L]and[(492.5±28.1) ng/L and (484.0±28.1) ng/L]. Compared with S2group, the levels of TNF-a and IL-6in lungs of S3group[(87.36±8.37) ng/L] and[(612.4±22.0) ng/L] showed a lower value, but still higher than those in S1group, all P<0.05.In lungs, the mRNA expression of RORyt and IL-17in S1and S2groups [(25.50±3.75) and (43.10±5.76)] and [(25.50±3.03) and (43.40±5.38)] were higher than those in C1and C2groups [(10.00±4.83) and (11.20±7.35)] and [(12.70±5.12) and (8.30±6.07)]. Compared with S2group, the mRNA expression of RORyt and IL-17in S3group [(37.70±5.36)] and [(37.60±5.13)] showed a lower value, but still higher than those in S1group, all P<0.05. In lungs, the percentage of Th17in S1and S2groups [(3.27±1.12)%和(7.19±2.24)%] were significantly increased than that in C1and C2groups [(1.80±0.75)%和(1.99±0.59)%]. Compared with S2group, the percentage of Th17in S3group [(4.57±1.15)%] showed a lower value, but still higher than that in S1group, all P<0.05. In lungs, the frequency of Th7cells, the mRNA expression of RORyt and IL-17in S1,S2and S3groups all had positive correlations with Lm and DI (r=0.709and0.674,0.802and0.812,0.705and0.766, all P<0.05).Conclusions Lung interleukin-17-producing CD4+T helper lymphocytes remains increasing on Cigarette Smoke-induced mice despite12weeks’ smoking cessation, suggesting that the up-regulation of Th17cells may be involved in the emphysema, and inflammation persistenting after smoking cessation.更多还原