短期香烟烟雾及脂多糖气道刺激对小鼠气道免疫细胞的影响

Effects of short-term stimulation of cigarette smoke and lipopolysaccharide on immune cells in airway of mice

目的:探讨香烟烟雾及脂多糖(LPS)短期刺激对小鼠气道免疫细胞的影响。方法:LPS组小鼠气管内(一次性)滴注10μg LPS,熏烟组小鼠每天熏9支香烟持续熏4 d,观测不同干预措施对小鼠体重的影响;检测支气管肺泡灌洗液(BALF)总细胞数及细胞分类计数,观察细胞形态,并利用PDB(2-丁酸佛波醇酯)诱导BALF中的细胞检测ROS产出率;荧光定量PCR法检测肺组织中GM-CSF和CXCL15 m RNA表达。结果:与实验前相比,对照组及LPS组小鼠体重未见明显变化(P>0. 05),熏烟组小鼠体重减少了18. 9%,其变化差异明显高于对照组和LPS组(P<0. 01)。与对照组相比,熏烟组小鼠气道灌洗液细胞总数和各类细胞总数均无明显变化(P>0. 05); LPS组小鼠气道灌洗液细胞总数、巨噬细胞计数和中性粒细胞计数均高于对照组和熏烟组(P<0. 01),且LPS组灌洗液巨噬细胞体积较大,形态不规则,中性粒细胞胞核分叶较对照组多。LPS组小鼠气道灌洗液细胞在PDB的诱导下及没有PDB的诱导下均比对照组和熏烟组具有更高ROS产出率(P<0. 01)。与对照组相比,LPS组小鼠肺组织中GM-CSF表达显著增高(P<0. 01),但CXCL-15和熏烟组小鼠肺组织中GM-CSF及CXCL-15的表达并未发生改变(P>0. 05)。结论:短期香烟烟雾刺激明显引起小鼠体重下降,但未能诱发明显气道炎症反应; 10μg LPS气道滴注可通过增加肺组织GM-CSF的表达,增加中性粒细胞的成熟和募集,增加中性粒细胞内氧化应激反应及产生ROS的能力,诱发明显气道炎症反应。

Objective:To explore the effects of the short-term stimulation of cigarette smoke and Lipopolysaccharide(LPS)on the immune cells in mouse airway.Methods:LPS(10μg)was given by intratracheal instillation in LPS group,and in smoke group,mice were exposed to nine cigarettes per day for 4 days.Body weight was measured before and after culling.BAL(Bronchoalveolar lavage)cells were stained with HE and then morphology of the BAL cells was observed and total cell count and cell differentials were measured.Production of ROS of BAL cells were measured with the L0-12 assay.GM-CSF and CXCL15 expression in lung tissue were detected by Real-time PCR.Results:The mice in the smoke group lost 18.9%body weight after 4 day smoke,and the variation was significantly higher than that of the control group and the LPS group(P<0.01).The total number of cells and cell differentials in BAL in the smoke group were not significantly increased compared to that in sham group(P>0.05).And LPS increased the number of total cells,macrophages and neutrophils(P<0.01).In the airway of mice treated with LPS,the macrophages were with large size and irregular shape,the nucleus of neutrophils were with more lobes than that in the control group.With or without the stimulation of PDB,inflammatory cells of BAL in the LPS group produced more ROS than that in sham group(P<0.01).Compared to control group,the expression of GM-CSF in the lung tissue of LPS treated mice was significantly higher(P<0.01),but the expression of CXCL-15 in the LPS group was unchanged,the GM-CSF and CXCL-15 in lung tissue of smoke mice were also unchanged(P>0.05).Conclusion:The short-term cigarette smoke stimulation obviously alleviates body weight,but fails to induce airway inflammatory obviously.Intratracheal instillation of LPS can increase the expression of GM-CSF in the lung tissue,recruit the neutrophils to the airway,and improve the ability of BAL cells to generate ROS,so LPS causes acute inflammation in the airway.