PM2.5影响微丝重排加剧慢性阻塞性肺疾病肺泡巨噬细胞吞噬缺陷的机制研究

The ambient fine particulate matter of less than or equal to 2.5μm in aerodynamic diameter (PM2.5) aggravates the deficiency in the alveolar macrophage (AM) phagocytosis in chronic obstructive pulmonary disease (COPD), which is correlated with frequent exacerbation of COPD. However, the underlying mechanism remains unclear. Microfilament rearrangement has been proved previously as the key factor in macrophage phagocytosis. Here, we propose that PM2.5 aggravates the deficiency in AM phagocytosis of bacterial pathogens in COPD, potentially through abnormal rearrangement of cytoskeleton regulated by TLR2,4/MyD88/PI3K/mTOR/Rho GTPases pathway. We will first silence TLR2, TLR4，PI3K and mTOR genes by RNA interference (RNAi) in RAW264.7 cells, a commonly used model cell line. The efficiency of gene silencing will be assessed at mRNA expression level, protein expression level and enzyme activity level, using quantitative PCR (qPCR), Western blot and GST-pull down assays. The effect of the gene silencing in microfilament reorganization and actin polymerization will be observed by the fluorescence confocal microscope and the effect in phagocytosis will be assessed by flow cytometry. We will then confirm our observation in vivo with gene knockout rats. The proposed experiments will improve our understanding of the mechanism underlying aggravated AM phagocytosis deficiency in COPD by PM2.5. Our research will shed light on the pathogenesis of the COPD exacerbations after the exposure in PM2.5.

PM2.5加剧慢性阻塞性肺疾病(慢阻肺)肺泡巨噬细胞(AM)吞噬功能缺陷，与慢阻肺急性加重密切相关，机制不清。研究证实微丝重排是影响AM吞噬的关键。本研究提出PM2.5通过TLR2、TLR4/MyD88/PI3K/mTOR/Rho GTPases通路，使AM微丝异常重排，加剧慢阻肺AM吞噬缺陷的假说。以RAW264.7细胞系为研究对象，用RNAi干扰TLR2、TLR4、PI3K、mTOR，用激光共聚焦显微镜观察干扰前后微丝形态、肌动蛋白的聚合，用流式细胞术、qPCR、蛋白印迹、GST-pull down等检测TLR2、TLR4、PI3K、mTOR和Rho GTPases家族RhoA,Racl,Cdc42的表达、活性变化及与吞噬的关系。再以基因敲除鼠为模型，在体内验证PM2.5通过该信号通路影响AM微丝重排，降低其吞噬功能的假说，探讨PM2.5致慢阻肺急性加重的分子机制。

本项目提出PM2.5通过TLR2、TLR4/MyD88/PI3K/mTOR/Rho GTPases通路，使肺泡巨噬细胞（AM）微丝异常重排，加剧慢性阻塞性肺疾病（慢阻肺）AM吞噬缺陷的假说。项目以慢阻肺小鼠和小鼠巨噬细胞系RAW264.7为研究对象，用PM2.5干预慢阻肺小鼠和RAW264.7细胞，流式细胞术检测AM和RAW264.7细胞吞噬能力，激光共聚焦显微镜观察细胞骨架变化，电镜扫描观察AM形态，qRCR、WB检测TLR4、PI3K、mTOR、Rho GTPases家族（RhoA，Rac1，Cdc42）、Arp2/3复合体、F-肌动蛋白的表达，小G蛋白活性试剂盒检测RhoA、Cdc42活性，从而阐明慢阻肺AM吞噬功能缺陷的可能机制，并探讨了PM2.5加剧慢阻肺AM吞噬功能缺陷及PM2.5通过影响Notch信号通路加重慢阻肺小鼠免疫紊乱的机制，研究党参多糖和黄芪多糖对PM2.5致慢阻肺小鼠AM吞噬功能障碍的保护作用。本研究的成果是⑴成功建立了短程单纯香烟烟雾暴露法制造慢阻肺小鼠模型及小鼠肺功能检测的方法；⑵探索出小鼠PM2.5雾化吸入的方法；⑶RNAi转染RAW264.7细胞系的方法；⑷揭示了慢阻肺AM吞噬功能低下的发生机制以及PM2.5对AM吞噬功能影响的机制，与TLR2、TLR4/MyD88/PI3K/mTOR/Rho GTPases通路调控致细胞骨架重排有关；⑸探索出甘肃省地道中药材党参黄芪对AM吞噬功能的保护作用；⑹在实验研究的基础上，进一步归纳总结了慢阻肺患者的相关临床资料。已发表论文16篇，在SCI 2区Environmental Pollution发表论著1篇，在Environmental Toxicology and Pharmacology、Brazilian Journal of Medical and Biological Research发表论著2篇，在《中华结核和呼吸杂志》、《中华医学杂志》发表论著6篇、综述1篇，在《中国实用内科杂志》《中国循证医学杂志》《中国呼吸与危重监护杂志》发表论著3篇，《护理学杂志》论著1篇，《国际呼吸杂志》综述2篇。该系列研究成果获2017年度甘肃省医学科技奖一等奖，2019年度甘肃省科技进步奖二等奖。培养博士研究生2名，硕士研究生12名。