慢性阻塞性肺疾病大鼠模型肺泡巨噬细胞炎症及调控机制探讨

目的探讨肺泡巨噬细胞在烟雾暴露致慢性阻塞性肺疾病（COPD）大鼠模型气道炎症中的作用及可能的调控机制。方法 12只Wistar大鼠随机分为对照组和COPD组。在COPD组以烟熏＋气道内滴注内毒素的方法建立COPD大鼠模型。行支气管肺泡灌洗液（BALF）细胞分类计数,分离大鼠肺泡巨噬细胞并用免疫荧光法进行鉴定,运用Western blot技术检测肺泡巨噬细胞胞浆和胞核NF-κB p65亚基的表达,ELISA法检测细胞培养上清TNF-α、MIP-2及IL-10浓度。结果 COPD组支气管炎症评分、平均内衬间隔显著高于对照组4.33±1.16比1.33±0.58,P=0.016;（168.77±11.35）μm比（93.61±4.16）μm,P=0.000]。COPD组BALF中细胞总数较对照组显著升高（P〈0.05）,分类以肺泡巨噬细胞及中性粒细胞为主（72.0±2.2）%和（18.3±8.3）%];COPD组肺泡巨噬细胞胞浆NF-κB p65蛋白的表达水平显著低于对照组,而胞核的表达水平则显著高于对照组（P〈0.05）;COPD组肺泡巨噬细胞培养上清中TNF-α、MIP-2的浓度显著高于对照组（P〈0.05）,IL-10浓度在两组间无统计学差异（P〉0.05）。结论以烟熏加气道内滴注内毒素的方法可成功建立COPD大鼠模型,表现为以巨噬细胞为主的慢性炎症,分泌TNF-α、MIP-2增加,其机制与NF-κB p65活化密切相关。

The Roll of Alveolar Macrophages in Airway Inflammation of COPD Rats

Objective To investigate the role of alveolar macrophages(AMs) in airway inflammation of smoke-induced COPD rat model and its possible regulating mechanism.Methods Twelve Wistar rats were randomly divided into a COPD group and a control group.The rat model of COPD was established with smoke exposure and LPS intrathacheal instillation.Bronchoalveolar lavage fluid(BALF) was collected for measurement of total and differential cell counts.Then AMs were isolated and identified by immunofluorescence.Western blot was employed to analyze the cytoplasmic and nuclear NF-κB p65 expression of AMs.The concentrations of TNF-α,macrophage inflammatory protein 2(MIP-2) and IL-10 in cell culture supernatant were assayed by ELISA.Results The scores of bronchitis and mean liner intercepts in the COPD group were significantly higher than those in the control group 4.33±1.16 vs.1.33±0.58,P=0.016;(168.77±11.35)μm vs.(93.61±4.16)μm,P=0.000)].The total cell count in BALF of the COPD group was significantly higher than that in the control group(P<0.05),and the AMs and neutrophils were predominant (72.00±2.22)% and(18.29±8.34)%].The cytoplasmic NF-κB p65 expression of AMs in the COPD group was significantly lower,while the nuclear NF-κB p65 expression was significantly higher(P<0.05) compared with the control group.The ELISA results showed that the concentrations of TNF-α and MIP-2 in culture supernatant of AMs in the COPD group were significantly higher than those in the control group(P<0.05),while the concentration of IL-10 was not significantly different between the two groups(P>0.05).Conclusions COPD rat model was established successfully with smoke exposure and LPS intratracheal instillation with a profile of macrophage-based chronic inflammation and increased secretion of TNF-α and MIP-2.The mechanism is closely related to activation of NF-κB.