煤工尘肺患者肺灌洗液中肺泡巨噬细胞自噬活动的研究

The study of autophagy in alveolar macrophages of patients with coal workers＇ pneumoconiosis

目的 比较不同期别煤工尘肺患者肺泡巨噬细胞自噬活动的差别.方法 对116例行全肺灌洗的观察对象和煤工尘肺患者进行现场调查,并收集其肺灌洗液,纯化后得到肺巨噬细胞,用透射电镜观察其自噬形态,采用免疫印迹方法检测自噬标志性蛋白LC3及自噬调控蛋白beclin1、mTOR和p-mTOR的表达量,分析接尘观察对象、Ⅰ、Ⅱ、Ⅲ期煤工尘肺患者肺泡巨噬细胞自噬活动的强弱变化.结果 透射电镜观察,发现自噬活动在不同期别煤工尘肺患者肺泡巨噬细胞中Ⅱ期煤工尘肺肺泡巨噬细胞中有自噬溶酶体明显多于观察对象和Ⅰ期煤工尘肺,Ⅱ期煤工尘肺的肺泡巨噬细胞中自噬体和自噬溶酶体明显少于Ⅱ期煤工尘肺.Ⅱ期煤工尘肺患者肺巨噬细胞LC3Ⅱ/Ⅰ和Beclin1蛋白表达量明显高于Ⅰ期,Ⅲ期煤工尘肺患者肺巨噬细胞LC3Ⅱ/Ⅰ和Beclin1蛋白表达量明显低于Ⅱ期,Ⅲ期煤工尘肺患者肺巨噬细胞LC3Ⅱ/Ⅰ蛋白表达量明显高于Ⅰ期,Ⅱ期煤工尘肺患者Beclin1蛋白表达量高于接尘观察对象,差异均有统计学意义（P＜0.05）.Ⅱ期煤工尘肺患者肺泡巨噬细胞mTOR和p-mTOR的表达量明显低于接尘观察对象和Ⅰ期,Ⅲ期尘肺患者肺泡巨噬细胞mTOR和p-mTOR表达量明显高于Ⅱ期,差异均有统计学意义（P＜0.05）.结论 不同期别煤工尘肺患者肺泡巨噬细胞自噬活动不同.

Objective To evaluate the differences in the autophagy activity of alveolar macrophages between patients with different stages of coal workers＇ pneumoconiosis （CWP）.Methods A total of 116 coal workers were investigated in the field.Their lung lavage fluid was collected and purified to obtain alveolar macrophages.The morphological characteristics of autophagy were observed by transmission electron microscopy.The expression of autophagy marker （LC3） and autophagy regulators （Beclin 1,mTOR,and pmTOR） was measured by Western blot.The autophagy activity of alveolar macrophages was compared between dust-exposed subjects and patients with stage Ⅰ,Ⅱ,and Ⅲ CWP.Results The autophagy activity of alveolar macrophages differed between patients with different stages of CWP,according to transmission electron microscopy.Patients with stage Ⅱ CWP had significantly higher protein expression of LC3 Ⅱ/Ⅰ and Beclin1 in pulmonary macrophages than those with stage Ⅰ CWP （P〈0.05）; patients with stage Ⅲ CWP had significantly lower protein expression of LC3 Ⅱ/Ⅰ and Beclin 1 in pulmonary macrophages than those with stage Ⅱ CWP （P〈0.05）,but had significantly higher protein expression of LC3 Ⅱ/Ⅰ and Beclin 1 than those with stage Ⅰ CWP （P〈0.05）;patients with stage Ⅱ CWP had a significantly higher protein expression of Beclin1 than the dust-exposed subjects （P〈0.05）.Patients with stage Ⅱ CWP had significantly lower expression of mTOR and p-mTOR in pulmonary macrophages than the dust-exposed subjects and those with stage Ⅰ CWP （P〈0.05）,while patients with stage Ⅲ CWP had significantly higher expression of mTOR and p-mTOR than those with stage Ⅱ CWP （P〈0.05）.Conclusion The autophagy activity of alveolar macrophages varies between patients with different stages of CWP.