慢性阻塞性肺疾病患者肺组织中自噬相关基因和蛋白的表达

Expression of autophage related genes and proteins in lung tissue of patients with chronic obstructive pulmonary disease

目的探讨慢性阻塞性肺疾病(COPD)患者肺组织标本中自噬相关基因和蛋白的表达情况。方法标本取自45例因肺部占位性病变行肺叶切除术患者的肺组织,根据患者有无COPD,分为非COPD组(对照组,15例)和COPD组(30例);再根据COPD患者的肺功能,将COPD组分为轻度COPD组(12例)、中度COPD组(12例)、重度COPD组(6例)。采用免疫组织化学染色观察肺组织中自噬蛋白微管相关蛋白1轻链3(LC3)B的表达;采用Western印迹法和实时定量PCR检测肺组织标本中自噬相关基因Atg5、酵母自噬相关基因Atg6同系物(Beclin1),以及自噬相关基因Atg5、Beclin1、LC3B的蛋白表达。结果非COPD组的肺组织结构清晰,未见支气管、肺泡结构破坏,炎性细胞浸润等。COPD组的肺组织结构破坏明显,肺气肿形成,肺实质有肺泡管、肺泡腔扩大,上皮细胞脱落、坏死等。LC3B蛋白主要表达于肺上皮细胞细胞质中,可见清晰的棕黄色颗粒。与非COPD组相比,COPD组肺上皮细胞细胞质中棕黄色颗粒增多,染色强度增加。轻度COPD组、中度COPD组和重度COPD组的Beclin1 mRNA均显著高于非COPD组(P值分别<0.01、0.05),轻度COPD组和中度COPD组的Atg5 mRNA均显著高于非COPD组(P值分别<0.01、0.05),重度COPD组与非COPD组间Atg5 mRNA的差异无统计学意义(P>0.05)。中度COPD组的Beclin1 mRNA和Atg5 mRNA分别显著高于轻度COPD组和重度COPD组(P值均<0.01),轻度COPD组的Beclin1 mRNA和Atg5 mRNA分别显著高于重度COPD组(P值均<0.05)。轻度COPD组和中度COPD组的Atg5蛋白表达量均显著高于非COPD组(P值分别<0.01、0.05),重度COPD组与非COPD组间Atg5蛋白表达量的差异无统计学意义(P>0.05);轻度COPD组、中度COPD组和重度COPD组的Beclin1蛋白和LC3B蛋白表达量均显著高于非COPD组(P值分别<0.01、0.05)。中度COPD组的Beclin1、Atg5、LC3B蛋白表达量分别显著高于轻度COPD组和重度COPD组(P值分别<0.05、0.01),轻度COPD组的Beclin1、Atg5、LC3B蛋白表达量分别显著高于重度COPD组(P值均<0.05)。结论在COPD的进展过程中自噬相关基因和蛋白表达增多,提示可能与疾病的发展有关。

Objective To investigate the expression of autophage-related genes and proteins in the lung tissue of patients with chronic obstructive pulmonary disease （COPD）. Methods Pulmonary tissues were obtained from 45 subjects who were undergoing single or bilateral Iobectomy or wedge resection for lung space- occupying lesions. They were divided into non-COPD group （n = 15）, mild COPD group （n = 12）, moderate COPD group （n = 12） and severe COPD group （n = 6） according to preoperative pulmonary function. The expression of autophagy protein LO3B was measured by immunohistochemical staining. The expressions of autophagy genes Atg5, Beclinl and autophagy proteins AtgS, Beclinl and LC3B in lung tissue were measured by Western blot and real-time polymerase chain reaction（RT-PCR）. Results In the non-COPD group, lung tissue was normal, and no bronchialor alveolar structure damage, inflammatory cell infiltration was found. In COPD groups, severe structural damage and emphysema were seen~ alveolar ducts and cavities were enlarged there were a large amount of the fall-off and necrosis of epithelial cells. LC3S protein was mainly expressed in the cytoplasm of pulmonary epithelial cells, and brown particles could be clearly seen. RT-POR analysis showed that the expression of Beclinl in the three COPD groups were significantly higher than that in the non-OOPD group（P〈0.01, 0.05）. The mRNA levels of Atg5 in mild and moderate COPD groups were significantly higher than that in the non-COPD group （P〈0.01, 0.05）, but there was no significant difference between severe COPD group and non-OOPD group （P〉0.05）. The levels of Atg5 and Beclinl mRNA in moderate COPD group were significantly higher than those in mild and severe COPD groups （all P〈0.01）, and Atg5 and Beclinl mRNA levels in mild COPD group were Significantly higher than those in severe COPD group （all P〈0.05）. Western blot analysis showed＇that the expression of Beclinl and LO3B in the three COPD groups were significantly higher than those in the non-OOPD group （P〈0.01, 0.05）. The protein levels of Atg5 in mild and moderate COPD groups were significantly higher than that in the n0n-OOPD group （P〈0.01, 0.05）, but there was no significant difference between severe COPD group and non-OOPD group （P〉 0.05）. The expression of Beclinl, Atg5 and LO3B proteins in moderate COPD group were significantly higher than those in mild and severe COPD groups （P〈0.05, 0.01）. The expression of Beclinl, Atg5 and LC3B proteins in mild COPD group were also significantly higher than those in severe COPD group （all P〈0.05）. Conclusion Autophagy genes and proteins are increased in the development of chronic obstructive pulmonary disease, indicating that autophegy may play an important role in the progress of COPD.