摘要
目的研究在慢性阻塞性肺疾病(简称慢阻肺)大鼠肺组织中自噬相关蛋白的变化。方法单纯烟熏法构建慢阻肺大鼠动物模型,采用Real time PCR,Western blot技术检测检测大鼠肺组织中PI3K、AKT、p-AKT、m TOR、p-m TOR及自噬相关基因LC3Ⅱ/Ⅰ、Atg5、Beclin1、P62、Atg7、Atg12的mRNA及蛋白表达,探讨在慢阻肺大鼠肺组织中自噬水平及自噬相关蛋白的变化。用LC3B免疫组化比较COPD组与正常大鼠间自噬水平的变化。结果 Real time PCR分析结果显示,与正常组相比,慢阻肺组大鼠肺组织中自噬相关基因Beclin1、Atg5、Atg12的mRNA表达显著增高(P<0.05,其中Beclin1、Atg5两组比较P<0.01)。Atg7的mRNA表达在慢阻肺组与正常组之间差异无统计学意义(P>0.05)。Western blot分析结果显示,慢阻肺组大鼠肺组织中PI3K蛋白、p-AKT/AKT及p-m TOR/m TOR蛋白表达显著降低(P<0.05)。自噬相关基因LC3Ⅱ/Ⅰ、Atg5、Beclin1蛋白表达增高(P<0.05,其中LC3Ⅱ/Ⅰ、Atg5两组比较P<0.01)。P62蛋白表达显著下降(P<0.01)。LC3B免疫组化显示,慢阻肺组LC3B表达高于正常组。结论烟熏12周的慢阻肺大鼠与正常组相比,自噬通路上游蛋白PI3K、p-AKT/AKT、p-m TOR/m TOR的表达降低,自噬蛋白表达增加,自噬水平增加。
Objective To investigate the changes of autophage-related protein in lung tissues of rats with chronic obstructive pulmonary disease （COPD）. Methods Passive cigarette smoking was used to establish COPD model in rats. The mRNA and protein expressions of PI3 K,total AKT, phosphorylated-AKT, total mTOR,phosphorylated-mTOR, and autophagy-related genes including LC3 Ⅱ / Ⅰ , Beclin1, Atg5, Atg7, Atg12,P62 in lung tissues were measured by real-time PCR and Western blot. The autophagy level was compared between the COPD rats and the normal rats by LC3B immunohistochemistry. Results Real-time PCR analysis showed that the mRNA expressions of Beclinl ,Atg5 and Atg12 significantly increased in lung tissues of the COPD rats compared with the normal rats （ all P 〈 0.05）. There was no significant difference between the COPD rats and the normal rats as for Atg7 mRNA expression （ P 〉 0.05）. Western blot analysis showed that the protein expressions of PI3K, p-AKT/AKT and p-mTOR/mTOR significantly decreased, the protein expressions of LC3 Ⅱ/Ⅰ ,Atg5 ,and Beclinl increased,and protein expression of P62 significantly decreased in lung tissues of the COPD rats compared with the normal rats （ all P 〈 0. 05 ）. LC3B immunohistochemistry showed that the LC3B expression was higher in the COPD rats than that in the normal rats. Conclusion The level of autophagy significantly increases in COPD rats with decreased expression of upstream proteins in autophagy signal pathway and increased expression of autophage proteins.
出处
《中国呼吸与危重监护杂志》
CAS
北大核心
2016年第5期470-476,共7页
Chinese Journal of Respiratory and Critical Care Medicine
基金
上海市自然科学基金(编号:14ZR1426800)
关键词
慢性阻塞性肺疾病
自噬
大鼠
肺组织
Chronic obstructive pulmonary disease
Autophagy
Rats
Lung tissue
