摘要目的 探讨细颗粒物(PM2.5)通过肌动蛋白相关蛋白(Arp)2/3复合体,影响慢性阻塞性肺疾病(慢阻肺)小鼠肺泡巨噬细胞(AM)吞噬功能的机制.方法 40只8周龄SPF级雄性BALB/c小鼠按随机数字表法分为健康对照组、健康PM2.5组、慢阻肺对照组、慢阻肺PM2.5组,每组10只.慢阻肺对照组及慢阻肺PM2.5组采用烟草烟雾暴露法建立慢阻肺小鼠模型,并称慢阻肺组,健康PM2.5组及慢阻肺PM2.5组小鼠连续雾化吸入PM2.5(662 μg/m3)90 d.流式细胞术检测AM吞噬异硫氰酸荧光素标记大肠杆菌(FITC-E.coli)的能力,用平均荧光强度(MFI)和吞噬阳性细胞百分比(吞噬％)表示;蛋白印迹法检测AM Arp2、F-肌动蛋白的含量;激光共聚焦显微镜检测AM Arp2、F-肌动蛋白、吞噬的FITC-E.coli的平均光密度及Arp2和F-肌动蛋白的共定位;扫描电镜观察AM吞噬FITC-E.coli后的超微形态.结果 慢阻肺对照组MFI和吞噬％[4 656±251,(31.9±1.7)％]低于健康对照组[8 657±247、(65.7±1.9)％,均P＜0.01];健康PM2.5组[7 653±228,(47.9±1.6)％]和慢阻肺PM2.5组[3 660±237、(19.2±1.2)％]均分别低于各自对照组(均P＜0.01),且慢阻肺组降低更明显.慢阻肺对照组AMArp2和F-肌动蛋白含量[(0.51±0.02)、(0.46±0.03)]低于健康对照组[(0.81±0.04)、(0.71±0.04),均P＜0.01];健康PM2.5组[(0.64±0.03)、(0.56±0.04)]和慢阻肺PM2.5组[(0.29±0.02、0.26±0.02)]均分别低于各自对照组(均P＜0.01),且慢阻肺组降低更明显.慢阻肺对照组Arp2、F-肌动蛋白及吞噬的FITC-E.coli平均光密度值分别为33.0±2.3、62.0±0.7和41.0±0.4,均低于健康对照组(141.0±4.2、145.0±2.9、189.0±2.6,均P＜0.01);健康PM2.5组(127.0±2.8、124.0±0.7、154.0±0.9)和慢阻肺PM2.5组(24.0±2.4、37.0±0.4、29.0±0.8)均分别低于各自对照组(均P＜0.01),且慢阻肺组降低更明显.AM Arp2和F-肌动蛋白共定位:慢阻肺组孟德斯共定位系数(MOC)为0.38±0.03,低于健康对照组(0.88±0.03,P＜0.01);健康PM2.5组(0.58±0.03)和慢阻肺PM2.5组(0.14±0.02)均分别低于各自对照组(均P＜0.01),且慢阻肺组降低更明显.健康对照组AM吞噬FITC-E.coli舌变形明显,细胞膜表面微皱褶多而高,微皱褶的游离缘有长而密集的丝状伪足伸出;慢阻肺组AM形态变化不明显,细胞膜表面的微皱褶稀少,丝状伪足伸出不良甚至缺失;健康PM2.5组AM形态变化轻微,多为不规则的圆形或椭圆形,细胞膜表面的微皱褶少而扁平,丝状伪足伸出短而少;慢阻肺PM2.5组AM形态僵硬,细胞膜表面的微皱褶极少而扁平,无明显丝状伪足伸出或突起.基础状态及PM2.5干预后,AM Arp2、F-肌动蛋白的含量及Arp2和F-肌动蛋白的MOC值均与MFI呈正相关.结论 慢阻肺小鼠AM吞噬功能低下,与Arp2/3复合体、F-肌动蛋白共同参与的细胞骨架异常重排有关;推测PM2.5可能通过抑制Arp2/3复合体、F-肌动蛋白共同参与的细胞骨架重排作用加剧慢阻肺小鼠AM吞噬功能下降.

abstractsObjective To investigate the mechanism of fine particulate matter (PM2.5) on the phagocytosis of alveolar macrophages (AM) in mice with chronic obstructive pulmonary disease (COPD) through actin-related protein (Arp) 2/3 complex.Methods Forty mice were divided into healthy control(A) group,healthy PM2.5 (B) group,COPD(C) group,and COPD PM2.5(D) group according to the random number table method.A mouse model of COPD was established by cigarette smoke exposure method.PM2.5 (662 μg/m3) model was established by continuously inhalation for 90 days in healthy PM2.5 group and COPD PM2.5 group.Flow cytometry was used to detect the ability of AM to phagocytose fluorescein isothiocyanate-labeled E.coli (FITC-E.coli),expressed as mean fluorescence intensity (MFI) and percentage of phagocytic positive cells (phagocytosis percentage);Western blotting was used to detect AM Arp2 and F-actin content,and laser confocal microscopy for AM Arp2 and F-actin and phagocytic FITC-E.coli average optical density and colocalization of Arp2 and F-actin,while scanning electron microscopy was used to observes the morphology of AM after phagocytizing FITC-E.coli.Results AM phagocytosis:MFI and phagocytosis percentage in the COPD group [4 656±251,(31.9± 1.7) ％] were lower than the healthy control group [8 657±247,(65.7±1.9) ％] (both P＜0.01);and healthy PM2.5 group and COPD PM2.5 group [7 653± 228,(47.9± 1.6)％ and 3 660±237,(19.2± 1.2)％] were lower than the respective control groups (all P＜0.01),and the decrease in the COPD group was more pronounced.AM Arp2,F-actin content:the COPD group (0.51±0.02,0.46±0.03) were lower than the healthy control group (0.81±0.04,0.71±0.04,both P＜0.01);the healthy PM2.5 group and the COPD PM2.5 group [(0.64±0.03,0.56±0.04) and (0.29±0.02,0.26±0.02)] were lower than the respective control groups (all P＜0.01),and the decrease in COPD group was more significant.Arp2,F-actin,and phagocytic FITC-E.coli mean optical density values:the COPD group (33.0±2.3,62.0± 0.7,41.0±0.4) were lower than the healthy control group (141.0±4.2,145.0±2.9,189.0±2.6,both P＜0.01);the healthy PM2.5 group and the COPD PM2.5 group (127.0±2.8,124.0±0.7,154.0±0.9,and 24.0±2.4,37.0±0.4,29.0±0.8) were lower than the respective control groups (all P＜0.01),and the decrease in the COPD group was more significant.Colocalization of AM Arp2 and F-actin:Montessori colocalization coefficient (MOC) (0.38±0.03) in the COPD group was lower than the healthy control group (0.88±0.03,P＜ 0.01);healthy PM2.5 group and COPD PM2.5 group [(0.58±0.03) and (0.14±0.02)] were lower than the respective control groups (both P＜0.01),and the decrease in COPD group was more significant.Morphology of AM phagocytosis of FITC-E.coli:AM in the healthy control group was obviously deformed,and the surface of the cell membrane was slightly wrinkled and high,and the free edge of the micro-pleated fold had a long and dense filamentous pseudopodia extension.The changes of morphology of AM in the COPD group was not obvious,the micro-wrinkles on the surface of the cell membrane were rare,and the filopodia poorly extended or even absent.The AM form of the healthy PM2.5 group changed slightly,mostly irregular circular or elliptical.The micro-wrinkles on the surface of the cell membrane were less and flat,and the filopodia protrudes short and less;the AM form of the COPD PM2.5 group was stiff,and the micro-wrinkles on the surface of the cell membrane were few and fiat,no obvious filopodia or protrusions.Correlation analysis:After basal state and PM2.5 intervention,AM Arp2,F-actin content and MOC values of Arp2 and F-actin were positively correlated with MFI.Conclusions The phagocytic function of AM in COPD mice was low,which was related to the abnormal rearrangement of cytoskeleton involved in Arp2/3 complex and F-actin.It was speculated that PM2.5 might inhibit Arp2/3 complex and F-actin.The cytoskeletal rearrangement of proteins was involved in the aggravation of AM phagocytosis in mice with COPD.